## LABORATORY QUALITY A **SSURANCE EVALUATION PROGRAM FOR** 

**ANALYSIS OF CRYPTOSPORIDIUM**  **UNDER THE SAFE DRINK ING**  **WATER ACT Information Collection Request:**  **Supporting Statement**

**U.S. ENVIRONMENTAL PROTECTION AGENCY Office of Ground Water and Drinking Water** 

**January 2014**

### ii **Table of Contents**
**1. Identification of the Information Collection**
................................ ................................ ........... 1 **1(a) Title of the Information Collection** 
................................ ................................ ...... 1 **1(b) Short Characterization** 
................................ ................................ ......................... 1 **2. Need For and Use of the Collection**
................................ ................................ ......................... 2 **2(a) Need/Authority for the Collection** 
................................ ................................ ....... 2 **2(b) Practical Utility/Users of the Data** 
................................ ................................ ....... 2 **3. Non -**  **duplication, Consultations, an d Other Collection Criteria** 
................................ .......... 2 **3(a) Non**  **- duplication** 
................................ ................................ ................................ ..... 2 **3(b) Public**  **Notice Required Prior to ICR Submission to OMB**
............................... 3 **3(c) Consultations** 
................................ ................................ ................................ ......... 3 **3(d) Effects of Less Frequent Collections** 
................................ ................................ ... 4 **3(e) General Guidelines** 
................................ ................................ ................................ 4 **3(f/g) Confidentiality/Sensitive Questions** 
................................ ................................ ..... 4 **4. The Respondents and the Information Requested**
................................ ................................ . 4 **4(a) Respondents/SIC Codes** 
................................ ................................ ........................ 4 **4(b) Information Requested** 
................................ ................................ ......................... 4 **5. The Information Collected -**  **Agency Activities, Collection Methodology, and Information Management** 
................................ ................................ ................................ ...................... 6 **5(a) Agency Activities** 
................................ ................................ ................................ ... 6 **5(b) Collection Methodology and Management** 
................................ ......................... 6 **5(c )**  **Small Entity Flexibility**
................................ ................................ ......................... 7 **5(d) Collection Schedule** 
................................ ................................ ............................... 8 **6. Estimating the Burden and Cost of the Collection**
................................ ................................ 8 **6(a) Estimating Respondent Burden** 
................................ ................................ ........... 8 **6(b) Estimating Respondent Costs** 
................................ ................................ ............ 11 **6(c) Estimating Agency Burden and Costs** 
................................ ............................... 13 **6(d) Estimating the Respondent**  **Universe and Total Burden and Costs**
............... 13 **6(e) Bottom Line Burden Hours and Cost Tables** 
................................ ................... 14 **6(f) Reasons for Change in Burden** 
................................ ................................ .......... 14 **6(g) Burden Statement** 
................................ ................................ ............................... 14

### iii

APPENDIX 

### A
................................ ................................ ................................ ................................ . A
- 1 Federal Register Notice : Published 2000 APPENDIX 

### B
................................ ................................ ................................ ................................ . B
- 1 Federal Register Notice : Published 2002 APPENDIX 

### C
................................ ................................ ................................ ................................ . C
- 1 Federal Register Notice : Published 2005 APPENDIX 

### D
................................ ................................ ................................ ................................ . D
- 1 Federal Register Notice : Published 2009 APPENDIX 

### E
................................ ................................ ................................ ................................ .. E
- 1 Federal Register Notice : Published XX XX, 2013 APPENDIX 

### F
................................ ................................ ................................ ................................ .. F
- 1 Comments on XX XX, 2013 Federal Register Notice APPENDIX 

### G
................................ ................................ ................................ ................................ . G
- 1 Application for Assessment of Cryptosporidium Laboratory QA APPENDIX 

### H
................................ ................................ ................................ ................................ . H
- 1 Positive Staining Control and OPR Slide Evaluation APPENDIX 

### I
................................ ................................ ................................ ................................ .... I
- 1 Example Report from Online Analy st Evaluation APPENDIX 

### J
................................ ................................ ................................ ................................ ... J
- 1 Method 1623 Ongoing Precision and Recovery (OPR) and Initial Precision and Recovery (IPR) Quality Control Criteria Calculated from Five Rounds of Proficiency Testing (PT) Data APPENDIX 

### K
................................ ................................ ................................ ................................ . K
- 1 Burden Tables

### 1

**Information Collection Request Section 1: Part A of the Supporting Statement** 
**1. Identification of the Information Collection 1(a)**  **Title of the Information Collection** Laboratory

Quality Assurance Evaluation Program for Anal ys is of Cryptosporidium under the Safe Drinking Water Act (Renewal) **OMB Number: 2040**  **- 0246**  **U.S. EPA**  **T racking Number**  **: 2067.0**  **4 1(b)**  **Short Characterization** The U.S. Environmental Protection Agency (EPA) is requesting a renewal of the information collection request (I CR) for the Laboratory Quality Assurance Evaluation Program for Anal ys is of Cryptosporidium under the Safe Drinking Water Act (Lab QA Program). This voluntary program applies to public and private laboratories that analyze water samples for Cryptosporidium . The program will help ensure that laboratories meet the quality assurance and the updated quality control criteria in Appendix J or EPA Method 162 3.1 ( EPA 816
- R
- 12
- 001, EPA Method 1623.1, January 2012 (PDF) when using EPA M ethods 1622 (EPA
- 815
- R
- 05
- 001, http://www.epa.gov/nerlcwww/documents/1622de05.pdf ) , 1623 (EPA
- 815
- R
- 05
- 002, http://www.epa.gov/nerlcwww/documents/1623de05.pdf ) , or 1623.1 for the determination of the identity and concentration of Cryptosporidium in source water b y filtration, immunomagnetic separation (IMS), and immunofluorescence assay (IFA) micro scop y. In formation collection activities required for laboratories seeking State Certification include: laboratory documents with resumes for each staff member; standard operating procedures (SOPs); Ongoing Precision and Recovery (OPR) control charts; Matrix Spike control charts; training records for all anal ys ts/technicians ; proficiency testing (PT) results; and a cop y of any certificates as documentation of equipment maintenance ; participating in off
- site evaluation activities, including submitting po sitive staining control and OPR slides and performing online analy st evaluation ; and participating in a 1 to 2 day on - site evaluation of laboratory

performance and data quality , including performing an independent count of a blind slide
. All materials are being collected b y participating S tate c ertification programs. This information collection will provide States with data to verify that the laboratories are capable of producing reliable data from the anal ys is of Cryptosporidium using EPA Method 1622 , EPA Method 1623 or EPA Method 1623.1
. The information collection will involve approximately 4 5 laboratories at a total cost of approximately

$ 5 90 , 858 .79 or 3504 labor hours annually . The total number of laboratories includes 45 laboratories that participate in the proficiency testing program. The estimated total State burden, including contractual costs, is estimated at $ 212,916.00 or 1968 labor hours annually , based on an estimate of 20 participating States (Appendix K)
. The estimated total

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Agency burden, in cluding contractual costs, is estimated at $ 125,170.15 or 1953 labor hours annually (Appendix K). **2. Need For and Use of the Collection 2(a)**  **Need/Authority for the Collection** The information collection is needed b y EPA to support the Cryptosporidium data gathering activities that are required under the Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR). The Lab QA Program is being renewed because the Cryptosporidium laboratory

evaluation program must continue for the duration of the LT2ESWTR
. Renewing the Lab QA Program will help ensure that qualified laboratories are available to drinking water plants. **2(b) Practical Utility/Users of the Data**  In formation collected under the Lab QA Program will be used by States and EPA to verify that Cryptosporidium occurrence data are generated b y qualified laboratories that can perform the anal ys es acceptably . U se of qualified laboratories for source water monitoring by drinking water utilities will help ensure that the data collected are of known and reliable quality
. Data quality could potentially be compromised in the absence of Laboratory Certification Programs . A list of laboratories that meet th e evaluation program criteria will be made available to the public by

States and/or EPA at http://water.epa.gov/lawsregs/rulesregs/sdwa/lt2/lab_home.cfm and will provide a resource to aid drinking water utilities (and others interested in monitoring water for Cryptosporidium occurrence for the protection of public health) in selecting a qualified anal ytical laboratory. **3. Non -**  **duplication, Consultations, and Other Collection Criteria**  **3(a) Non**  **- duplication**  The information requested from the respondents under this I CR is not available from other sources. The information requested will be used to assess the current ability of a laboratory to reliably

anal yz e Cryptosporidium in water using EPA Method 1622 , EPA Method 1623 or EPA Method 1623.1 . Information submitted for previous programs, such as the Information Collection Rule, is not applicable because older analy tical methods were used and quality control requirements were different. The determination that this information is not available from other sources was made b y the Office of Ground Water and Drinking Water Technical Support Center (TSC), which will be administering the Lab QA Program, and which has worked closel y since 1996 with the community of capable laboratories that will be affected by this information collection.

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**3(b) Public**  **Notice Required Prior to ICR Submission to OMB** On August XX , 20 13 (XX FR XXXX ), EPA sought comments on this I C R pursuant to 5 CFR 1320.8(d). EPA received XX# comments during the comment period, which are addressed in the I CR. C op ies of the previous four Federal Register notice s are attached in Appendi ces A , B, C, and D
. The ICR was last renewed in 200 9 (See Appendix D ). EPA has developed a webpage to provide further information on the pro gram. The website can be accessed at http://water.epa.gov/lawsregs/rulesregs/sdwa/lt2/lab_home.cfm
. **3(c) Consultations**  EPA conducted meetings with representatives of the drinking water treatment industry and the community

of laboratories expected to seek EPA recognition under the Laboratory Quality Assurance Evaluation Program in Cincinnati, OH, on January 23 and March 12
- 13, 2001, and in Washingto n, DC, on February 13
- 14, 2001. EPA presented and discussed draft plans for the laboratory

evaluation program at these meetings and sought input from the drinking water utility

and laboratory

representatives that attended these meetings. In addition, EPA h as been working with States and EPA Regions to integrate Cryptosporidium laboratory approval/oversight into existing State certification/accreditation programs. Participating States will approve and oversee laboratories that support the second round of LT2 ESWTR monitoring. L aborato ries were asked to estimate the loaded cost of legal, clerical, technical and management personnel per hour , and average cost for anal ys es of a sample using Method1623.1
. In addition, laboratories were asked to estimate the hours that each category of personnel would spend to complete the activities associated with submitting a laboratory evaluation application package, performing off

- site evaluation activities, preparing a nd hosting on
- site evaluation, and performing and reporting two sets of PT samples per year . L aboratories were also asked to estimate the operation and maintenance costs associated with PT samples and audits. Six laboratories have supplied burden estimate information for participating in a state laboratory certification program:

· Charlotte
- Mecklenberg Utilities

– Environmental L aboratory Services

· Pennsylvania Department of Environmental Protection, Bureau of Laboratories

· Environmental Science Corporation

· Bio Vir Laboratories, Inc.

· CH Diagnostic & Consulting Service, Inc.

· Underwriters Laboratories, Inc. States were asked to estimate the loaded cost of legal, clerical, expert, technical or certification officer, and management personnel per hour
. In addition, states were asked to estimate the hours that each category of personnel would need to complete the activities associated with reviewing a laboratory evaluation application package, conducting off
- site evaluation activities, conducting on
- site evaluations, notifying laboratories of certification status, reviewing and tracking PT samples, coordinating follow
- up activities for poor PT results and maintaining a list of certified laboratories on the State website
. Two states have supplied burden estimates for p roviding laboratory certification:

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· N ew J ersey

Department of Environmental Protection, Office of Quality Assurance

· Kentucky Department of Environmental Protection, Division of Water **3(d) Effects of Less Frequent Collections**  Under the Lab QA Program, laboratories anal yz e single
- blind proficiency test samples 2 times per year. This frequency enables States to independentl y verify that laboratories perform in an acceptable manner. Less frequent proficiency test samples may not sufficiently

capture a laboratory ’s performance over time. Laboratories report proficiency test results to the PT provider within 14 day s of sample receipt. Reporting proficiency test sample results at this frequency allows States to respond in a timely manner to any

problems the laboratory

may be having with anal ys is of Cryptosporidium in water. **3(e) General Guidelines**  The Lab QA Program adheres to all of OMB’s general guidelines for information collection. **3(f/g) Confidentiality/**  **Sensitive Questions** The Lab QA Program does not ask any confidential or sensitive questions. **4. The Respondents and the Information Requested 4(a)**  **Respondents/ NAICS**  **Codes** The following is a list of NAI CS codes associated with laboratories affected by the requirements of this ICR: 541380

– Professional, Scientific, and Technical Services: Testing Laboratories 9 24 11 0

– Public Administration: Administration of Air and Water Resource s and Solid Waste Management Programs **4(b) Information Requested**  (i) Data Items Data items requested from laboratories:

· La boratory evaluation application information

· Resumes for each staff member seeking EPA recognition under the program

· Standard operating procedures (SOPs)

· Ongoing precision and recovery (OPR) and matrix spike (MS) control charts

· Training records for all anal ys ts/technicians added since the last audit

· Data packages

· Documentation of off
- site evaluation activities , including submitting positive control and

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OPR slides and performing internet anal ys t evaluation

· P roficiency

testing (PT) data

· Documentation of corrective actions taken in response to any deficiencies noted during the on - site evaluation

· Documentation of corrective actions taken in response to poor PT results Laboratories and States m aintain:

· P roficiency T est data (ii) Respondent Activities Acti vities for laboratories seeking state certification :

· Completing laboratory evaluation application package (no more than 1 time per 2 ye ar period) (See Appendix G )

· Perform ing off

- site evaluation activities (no more than 1 time per 2 year period)

· Analyzing P roficiency T est samples (set of 3 samples, 2 times per year) and reporting P roficiency T est data

· Hosting on
- site evaluation , including independent count of a blind slide, (1 time per 2 or 3 ye ar period)

· Perform ing follow
- up action for poor PT results

· Analyzing and reporting P roficiency T est s for each additional method version (set of 3 samples, 2 times per year) Response Activities/Year /L aboratory = (2 2.5 application packages + 2 2.5 off
- site activiti es + 2 2.5 on - site evaluations + ( 45 labs* 2 PT s) + (4 labs with additional method versions* 2 PT s) + 7 PT failures /45 labs = 3. 8 Responses/year /lab Activities for States with Laboratory Certification Programs :

· 

### Reviewing laboratory evaluation application packages (1 time per laboratory per 2 year period)

· 

### Notifying laboratories of certification status and maintaining a list of certified laboratories on State website (1 time per laboratory per 2 ye ar period)

· 

### Conducting and reviewing off
- site evaluation activities (1 time per laboratory per 2 year period)

· 

### Co nducting on
- site evaluations of the laboratories seeking certification (1 time per laboratory

per 2 year period)

· 

### Tracking PT results and c oordinating follow
- up activities for poor PT results Response Activities/Year /State = (22.5 application packages + 22. 5 off
- site activities + 22.5 on
site evaluations + (45 labs*2 PT s) + (4 labs with additional method versions*2 PT s) + 7 PT failures)/20 states = 8.6 Responses/year /state

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**5. The Information Collected -**  **Agency Activities, Collection Methodology, and Informa tion**  **Management 5(a)**  **Agency Activities** Agency (EAP Regions and OGWDW) activities associated with the Lab QA Program consist of the following:

· Assisting State Certification Officers with review of laboratory evaluation application packages

· Assisting State Certification Officers with off
- site evaluation activities, including slide evaluation and online analy st evaluation

· Assisting State Certification Officers with on
- site evaluation activities , including independent count of blind slides

· Assisting State Certification Officers with interpretation of P T results

· Maintaining a list of links to State websites and/or certified laboratories on EPA’s website **5(b) Collection Methodology and Management**  **Laboratories Seeking State Certification**  L aboratories interested in obtaining S tate certificati on of their capability to perform anal ys es using EPA Method 1622, EPA Method 1623 or EPA Method 1623.1 and continuing to demonstrate, proficient and reliable detection and enumeration of Cryptosporidium in surface water sources for public water s ys tems may contact State personnel responsible for laboratory certification
. Certified laboratories are responsible for notifying the S tate of losses of key personnel or essential equipment and changes in policies or procedures that directly affect the validity of data or any other change affecting the capability of the laboratory, including change in laboratory location. Participating laboratories are to also demonstrate ongoing capability and method performance b y following all applicable method quality contro l (QC) procedures, anal yz ing P roficiency T esting (PT) samples (2 times per year), submitting requested data to the S tate, and participating in periodic evaluations. The Method procedures have been updated to reflect that the minimum recovery for Cryptosporidium in ongoing precision and recovery (OPR) samples is now 33 percent (See Appendix J ). Laboratories are to document a minimum of 33 percent recovery

for OPR samples in an updated QC chart prior to anal ys is of LT2ESWTR samples at the frequency

required in Section 9.7 of EPA method 1622/1623 and Section 9.8 of Method 1623.1. If a laboratory submits poor PT results, EPA may recommend additional follow
up action to demonstrate that the laboratory ’s p erformance remains acceptable. Additional actions may

inclu de submission of PT slides, repeat anal ys es, providing additional QC data, and investigation of problem s with reagents and equipment. Repeated failure to demonstrate laboratory

capability

and acceptable method performance may result in suspension or downgrading of approval status as outlined in 2009 Federal Register Notice (https://federalregister.gov/a/E9
- 4009 ) (See Appendix D).” EPA expects that State certification personnel , or their designee, will request a package with documentation of laboratory personnel status, equipment maintenance, standard operating procedures, training records, and QC charts. EPA expects that the State will evaluate the package for completeness , and would generall y fol low these steps :

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1. The laboratory

will send positive staining control and OPR slides for evaluation by State certification officer or their designee (Appendix H ). 2. The laboratory

will order on - si te audit package consisting of two vials containing 50 m L blind samples, 2 vials of artificial matrix, 2 IMS control samples, and 2 anal ys t evaluation slides. One slide is required per analyst and a back up slide. These will be used in presence of an auditor; therefore, on - site evaluation date should be within product expiration. Each anal ys t will perform an independent count of one slide. The results and anal ys t evaluation slides and associated examination forms will be submitted to a State , if requested
. 3. The laboratory will schedule an online anal ys t evaluation of performance for microscopists to demonstrate their ability to identify Cryptosporidium oocy sts . An example report from the internet anal ys t evaluation is shown in Appendix I. 4. The S tate will conduct one 2
- day on - site evaluation that will primarily focus on anal ys t skills and data recording. Laboratory personnel may be asked to prepare a fresh OPR and positive control for review during the on
- site evaluation. In addition, laboratory personnel will be asked to use the blind oocy st suspension s ordered in #2 for spiking reagent water and an IMS control in the presence of an auditor, and then complete the anal ys es within applicable method holding times and send results to the S tate (Appendix G ). 5. The State will send the laboratory a report a ft er the evaluation is complete. The laboratory is then asked to provide written responses to any deficien cies identified in the report. Provided all responses to the deficiencies cited in the report are acceptable, the State will then base its decision fo r laboratory certification on PT results, quality of the positive control and OPR slide, slide counts, online anal yst evaluation , on - site evaluation and recovery values for blind anal ys es initiated during the on
- site evaluation (See Appendices G, H, and I ). **5(c) Small Entity Flexibility**  The Lab QA Program is a voluntary program; any entity not interested in gaining approval to support LT2 ESWTR is not required to participate
. Small businesses are defined as any business that is independently owned and operated and not dominant in its field as defined b y the Small Business Administration (SBA) regulations under Section 3 of the Small Business Act. Small businesses may

opt to seek state recognition of laboratory capability to perform Cryptosporidium water analy ses using onl y one version of EPA Method 1622 /1623 or EPA Method 1623 .1 , as opposed to being evaluated for multiple versions, and reduce the burden associated with participation in the L ab QA Program.

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**5(d) Collection Schedule**  Laboratory

certification is voluntary
. Laboratories that have successfull y completed the audit process will not be evaluated more than once per two - ye ar period. TSC’s oversight of LT2ESWTR laboratories during Round 1 represented an exception to the traditional laboratory

certification/accreditation approach. At the time, labs and States had limited experience with Cryptosporidium monitoring. With the experience gained b y laboratories, as well as States, during the first round of monitoring, all parties should be more comfortable with the monitoring and laboratory requirements, thus enabling interested States to take a primary rol e in lab certification while EPA provides guidance and technical support. The approach for Round 2 creates consistency with the approach for approval/oversight of laboratories for all other regulated anal yt es and is provided for in the LT2ESWTR rule. A number of States have already expressed an interest in adding Cryptosporidium laboratory approval to their programs and have participated in TSC’s Cryptosporidium Laboratory Certification Officers Training Course in 2010, 2011 and 2013. **6. Estimating the Bu rden and Cost of the Collection**  **6(a) Estimating Respondent Burden**  Below are summaries of respondent burden hours for this information collection. E PA consulted with six respondents from the community of laboratories that have voluntarily applied for State certification to perform Cryptosporidium anal ys es using EPA Method 1622 , 1623 , and 1623.1, and 2 respondents from the community of States integrating certification programs to obtain burden hour estimates. For specific burden breakdowns, refer to the Participating Laboratories Seeking Certification and State Annual B urden tables in Appendix K . The burden estimates were also addressed in the Federal Register Notice published in July 2013 (See Appendix E ). Based o n the first round of LT2ESWTR monitoring, EPA estimates that 45 laboratories will participate in State Certification Program s , of which 15 are private and 3 0 are state or local government entities . EPA estimates that 20 States will develop programs to certify Cryptosporidium laboratories
. Twenty- one states have Cryptosporidium laboratories within their state boundaries and 19 states responded positively to an industry survey [ASDWA]. **Activities for Laboratories Seeking State Certification**  Laboratories seeking State certification for anal ys is of Cryptosporidium will complete an application package, including: laboratory documents with resumes for each staff member; standard operating procedures (SOPs); Ongoing Precision and Recovery charts; and Matrix Spike control charts; training records for all anal ysts/technicians; data package; proficiency testing (PT) results; and a cop y of any certificate s as documentati on of equipment maintenance. Since laboratories will submit the application no more than 1 time per 2 ye ar period, the number of laboratories expected to submit applications were evenl y distributed over a 2 year- period to

### 9

estimate burden hours and costs per ye ar (e.g., laboratories seeking c ertification , 45 laboratories/2 ye ars = approximately 22.5 labs/ye ar). Burden hours associated with submitting the completed application package for the laboratories appl yi ng for certification are estimated at 2 93 labor h ours per ye ar. Laboratories seeking certification will also complete off
- site evaluation activities including submitting positive staining control and OPR slides, and performing online anal ys t evaluation . Since laboratories have to complete off
- site ev aluation activities no more than 1 time per 2 year period, the number of laboratories expected to complete off
- site evaluation activities were evenl y distributed over a 2 ye ar- period to estimate burden hours per year (e.g., laboratories seeking c ertificati on , 45 laboratories/2 ye ars = approximately 22.5 labs/ye ar). Burden hours associated with completing off
- site evaluation activities for the laboratories applying for certification are estimated at 486 labor hours per year. Each laboratory

seeking State c ertification will participate in a 1 to 2 day on - site evaluation . The burden hours associated with this task include time required to attend short briefings b y the auditors before and after the audit, demonstrate the techniques for the methods for which they

are seeking certification , participate in discussions with th e auditors, and respond to any deficiencies noted in the audit report. Laboratories will purchase blind spikes and slides from a qualified vendor and will process samples to demonstrate method performance and perform an independent count of a blind slide
. Because laboratories will undergo an on
- site evaluation no more than 1 time every 2 ye ars, the number of laboratories expected to be evaluated were evenl y distributed over a 2 ye ar period to estimate burden hours per year (e.g., laboratories seeking c ertif ication , 45 laboratories/2 ye ars = approximately 22.5 labs/year). Burden hours for all laboratories appl yi ng for certification are estimated at 889 labor hours per ye ar (Appendix K). Laboratories will anal yz e a set of proficiency test samples, which may include confounding organisms (3 samples per set) 2 times a year for each method version for which they are seeking certification . The burden estimates associated with this task include labor associated with analyzing samples and documenting the data for each proficiency test set. Burden hours for all laboratories seeking c ertification and anal yz ing 1 set of proficiency test samples 2 times a year are estimated at 1 541 labor hours per ye ar (Appendix K). It is estimated that 4 laboratories seeking State certification will perform more than 1 method version and will perform an additional set of PT samples for each additional method version 2 times a year. Burden hours for laboratories seeking certification and performing an additional set of PT samples for each additional method version are estimated at 1 37 labor hours per year (Appendix K). EPA estimates that a small subset of laboratories seeking State certification will have to perform follow
- up activities based on inadequate QA/QC, failed OPRs, incomple te records, delayed communication to the S tate or poor PT results. Burden hours for laboratories seeking c ertification and performing follow
- up activities based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication or poor PT results are estimated at 1 58 labor hours per year (Appendix K).
**Activities for State**  **s Integrating Cryptosporidium**  **C ert** **ification P**  **rograms**

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States integrating certification programs for anal ysis of Cryptosporidium will review a laboratory ’s application package, including: laboratory documents with resumes for each staff member; standard operating procedures (SOPs); Ongoing Precision and Recovery charts; Matrix Spike control charts; training records for all anal ysts /technicians; data packages, proficiency testing (PT) results; and a cop y of any certificate s as documentation of equipment maintenance. Since laboratories will submit the application no more than 1 time per 2 year period, the number of laboratories expect ed to submit applications were evenl y distributed over a 2 year- period to estimate burden hours and costs per ye ar (e.g., laboratories seeking c ertification , 45 laboratories/2 ye ars = approximately 22.5 labs/ye ar). Burden hours associated with all States reviewing completed application package for the laboratories applying for certification are estimated at 416 labor hours per ye ar (Appendix K)
. States integrating certification programs will also review off
- site evaluation activities including positive staining control and OPR slides, and online anal ys t evaluation . Since laboratories have to complete off
- site evaluation activities no more than 1 time per 2 year period, the number of laboratories expected to complete off
- site evaluation activities were ev enl y distributed over a 2 ye ar- period to estimate burden hours per ye ar (e.g., laboratories seeking c ertification , 45 laboratories/2 years = approximately 22.5 labs/ye ar). Burden hours associated with all States reviewing off
- site evaluation activities for the laboratories applying for certification are estimated at 315 labor hours per year (Appendix K)
. States will conduct a 1 to 2 day on - site evaluation of each laboratory seeking State recognition of laboratory capability under the Lab QA Program . The burden hours associated with this task include time required to travel to and from the laboratory , conduct short briefings before and after the audit, observe the techniques (including independent count of a blind slide) for the methods for which the laboratory is seeking certification, and participate in discussions with the laboratory

personnel
. Because laboratories will undergo an on
- site evaluation no more than 1 time every

2 years, the number of laboratories expected to be evaluated were evenl y distributed over a 2 ye ar period to estimate burden hours per ye ar (e.g., laboratories seeking c erti fication , 45 laboratories/2 years = approximately 22.5 labs/ye ar). Burden hours for States conducting on
- site evaluations for laboratories applying for certification are estimated at 450 labor hours per ye ar (Appendix K). States integrating Cryptosporidium certification programs will track and review proficiency

test results (3 samples per set) 2 times a year for each method version for which the laboratory

is seeking certification. The burden estimates associated with this task include labor associated with receiving, documenting, tracking and reviewing each laboratory ’s PT results. Burden hours for States tracking and reviewing 1 set of proficiency test samples 2 times a year are estimated at 135 labor hours per ye ar (Appendix K). It is estimated that 4 laboratories seeking State certification will perform more than 1 method version and will perform an additional set of PT samples for each additional method version 2 times a year. Burden hours for States reviewing an additional set of PT samples for each additional method version are estimated at 12 labor hours per ye ar (Appendix K). EPA estimates that States will perform follow
- up activities with a small subset of laboratories seeking c ertification based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication to the state or poor PT results. Burden hours for States coordinating these follow
- up activities are estimated at 21 labor hours

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per year (Appendix K). States will notify

laboratories of certification status after satisfactory c ompletion of all activities. The burden hours associated with this task include time to produce an audit report noting any deficiencies, develop certificate s , document communication with the laboratory personnel , and maintain certified laboratory list on S tate website
. Because laboratories will be notified no more than 1 time every 2 years, the number of laboratories expected to be notified were evenl y distributed over a 2 ye ar period to estimate burden hours per ye ar (e.g., laboratories seeking certificati on , 45 laboratories/2 ye ars = approximately 22.5 labs/ye ar). Burden hours for States conducting the laboratory notifications and maintaining certified laboratory lists are estimated at 619 labor hours per ye ar (Appendix K). **6(b) Estimating Respondent**  **Costs** Below are summaries of respondent burden costs for this information collection. EPA consulted with fewer than nine respondents from the community of laboratories that have voluntarily

applied for EPA approval of laboratory capability

to perform Cryptosporidium anal ys es using EPA Method 1622 , 1623 , and 1623.1 and States integrating Cryptosporidium Certification Programs to obtain burden cost estimates. Respondent costs associated with anal ys is of PT samples include labor and O&M costs, which are estimated by the laboratories and include cost of anal ytical sample and PT set
. Respondent costs associated with evaluation activities include labor and O&M costs, which are estimated by the laboratories and include application fees and travel fees for auditors
. For s pecific burden breakdowns, refer to the Participating Laboratories Seeking C ertification and State Annual Burden tables in Appendix K
. EPA estimates that 45 laboratories will seek State Certification and 20 States will integrate Cryptosporidium into their existing Certification Programs
. **Cost for Laboratories Seeking**  **Certification** Burden costs associated with submitting the completed application package for the laboratories appl yi ng for certification are estimated at $ 113 , 056.8 8 per ye ar . Burden costs associated with this task include $ 24,856.88 for labor and $ 88,200 .00 for Operations and Maintenance (O&M) (Appendix K). Burden costs associated with completing off
- site evaluation activities for the laboratories are estimated at $ 56,846.25 per year. Burden costs associated with this task include $ 41,096.25 for labor and $ 15,750.00 for O&M (Appendix K). Burden costs for all laboratories participating in the one 2
- day on - site evaluation are estimated at $ 150,502.50 per year (Appendix K). Burden costs associated with this task include $ 77,287.50 for labor and $ 73,215.00 for O&M (Appendix K). Burden costs for all laboratories anal yz ing 1 set of PT samples 2 times a year are estimated at $ 228,127.50 per year. Burden costs associated with th is task include $ 128,002.50 for labor and $ 100,125.00 for O&M (Appendix K). It is estimated that 4 laboratories seeking

### 12

State certification will perform more than 1 method version and will perform an additional set of PT samples for each additional method version 2 times a year. Burden costs for laboratories performing an additional set of PT samples for each additional method version are estimated at $ 20,278.00 per ye ar. Burden costs associated with this task include $ 11,378.00 for labor and $ 8,900.00 for O&M (Appendix K). A small subset of laboratories seeking certification will have to perform follow
- up activities based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication to EPA or poor PT results. Burden costs for laboratories perf orming follow
- up activities based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication or poor PT results are estimated at $ 22,047.67 per ye ar. Burden costs associated with this task include $ 13,181.00 for labor and $ 8,866.67 for O&M (Appendix K). **Cost for State**  **s Integrating**  **Cryptosporidium Certification Programs**  Burden costs associated with reviewing the completed application package for the laboratories appl yi ng for certification are estimated at $ 45,123.75 per ye ar. Burden costs associated with this task include $ 45,123.75 for labor and $ 0.00 for Operations and Maintenance (O&M) (Appendix K). Burden costs associated with conducting and reviewing off
- site evaluation activities for the laboratories applying for certification are estimated at $ 34,290.00 per ye ar. Burden costs associated with this task include $ 34,290.00 for labor and $ 0.00 for O&M (Appendix K). Burden costs for all States conducting 1
- or 2
- day on - site evaluation are estimated at $ 48,735.00 per ye ar (Appendix K) . Burden costs associated with this task include $ 48,735.00 for labor and $ 0.00 for O&M (Appendix K). Burden costs for all States reviewing 1 set of PT samples 2 times a year are estimated at $ 14,445.00 per ye ar. Burden costs associated with this task include $ 14,445.00 for labor and $ 0.00 for O&M (Appendix K). It is estimated that States will review results from 4 laboratories seeking certification that perform more than 1 method version and will review an additional set of PT samples for each additional method version 2 times a year. Burden costs for States reviewing an additional set of PT samples for each additional method version are estimated at $ 1,284.00 per ye ar. Burden costs associated with this task include $ 1,284.00 for labor and $ 0.00 for O&M (Appendix K). States will conduct follow
- up activities with a small subset of laboratories seeking certification based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication or poor PT results. Burden costs for State s conducting the coordination based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication or poor PT results are estimated at $ 2,247.00 per ye ar. Burden costs associated with this task include $ 2,247.00 for labor and $ 0.00 for O&M (Appendix K). Burden costs for all States notifying laboratories of certification status after satisfactory completion of all activities and maintaining a list of certified laboratories on state website are estimated at $ 66,7 91.25 per year . Burden costs associated with this task include $ 66,791.25 for labor and $ 0.00 for O&M (Appendix K).

### 13

**6(c) Estimating Agency Burden and Costs**  Below are Agency

burden hours and associated fi nancial costs pertaining to implementation of the Lab QA Program. For a specific breakdown of burden hours and financial costs, refer to the Agency Annual Burden table in Appendix K . Costs and burden hours are broken out based on activities completed by th e Agency

and supporting contractors. EPA estimates an average hourly cost of $ 87.25 /hour for Agency program management staff. Based on the published schedule of contractor labor rates for the years covered by this program, the average loaded burden hours and costs for contractor labor were estimated at $ 62.60 /hour for management staff, and $ 43.57 /hour for technical staff. Agency burden is estimated based on the labor hours associated with performing each task for each laboratory seeking State certificati on . To get the total annual cost, hours and costs are then multiplied by

the estimated number of respondents and added to the capital and O& M costs. The burden associated with each information collection task is shown in a separate row of the burden table. EPA estimates that 45 laboratories will participate in the EPA L ab QA Program and 20 States will integrate Cryptosporidium Certification Program s . Twenty
- one states have Cryptosporidium laboratories within their state boundaries and 19 states responded positively to an industry

survey [ASDWA]. If needed, the Agency

will assist State Certification Officers with review of laboratory evaluatio n application packages. The Agency burden associated with this review is estimated at 461 labor hours and a cost of $ 25,224.75 per ye ar. If needed, the Agency

will assist State Certification Officers with off
- site evaluation activities, including interne t anal ys t evaluation , and positive control and ongoing precision and recovery (OPR) slide evaluation. The Agency burden associated with off
- site evaluation activities is estimated at 39 4 labor hours and a cost of $ 24,058.78 for labor and $ 1,775.00 for O&M per year . If needed, the Agency

will assist State Certification Officers with on
- site evaluation activities, including independent counts of blind slides by analy sts. The Agency

burden associated with on
- site evaluation activities is estimated at 416 la bor hours and a cost of $ 28,178.10 per ye ar. If needed, the Agency

will assist State Certification Officers with interpretation of PT results. The Agency

burden associated with this activity is estimated at 506 labor hours and a cost of $ 36,030.60 per year. The Agency will assist State Certification Officers with interpretation of PT results for additional method versions. The burden associated with this activity is estimated at 45 labor hours and a cost of $ 3,202.72 per ye ar. The Agency

will maintain a list of links to S tate websites and/or certified laboratories on EPA’s website. The Agency burden associated with this technical support is estimated at 130 labor hours and a cost of $ 8,475.20 per ye ar . **6(d) Estimating the Resp**  **ondent Universe and Total Burden and Costs**

### 14

The affected entities include public and private water testing laboratories , and public administrators of environmental protection programs . EPA estimates that 45 laboratories will seek certification , of which 15 are private and 30 are state or local government entities
. EPA estimates that 20 States will integrate Cryptosporidium into existing certification programs. The respondent to tal burden and cost are provided in the Total Respondent and Agency Burden t ables in Appendix K and are described in greater detail in Sections 6(a)
- 6(c). **6(e) Bottom Line Burden Hours and Cost Tables**  (i) Respondent Tall y Refer to the burden table in Appendix K titled, Total Respondent and Agency Burden , for a specific breakdown of the respondent costs. The Lab QA Program will affect approximately 65 respondents. The laboratory and State respondents will engage in 13 different tasks (refer to Section 4(b)(ii)) involving 5472 labor hours and costing approximately $ 508,718.13 per ye ar for labor (Appendix K) . Respondents will invest $0.00 per year in capital/start
- up costs and $ 2 95 , 056 .67 per ye ar in O&M costs (Appendix K)
. (ii) Agency

Tally Refer to the burden table in Appendix K titled, Total Agency and Agency Burden , for a summary of Agency

costs. Six Agenc y t asks are associated with the Lab QA Program. These tasks will involve approximately 1953 labor hours annually resulting in a cost of $ 123,395.15 per ye ar for labor. The Agency will invest approximately $0.00 per year in capital/start
- up costs and $ 1,775.00 per ye ar in O&M costs. **6(f) Reasons for Change in Burden**  Changes in burden have occurred due to inflation, re
- evaluation of hours for tasks, re evaluation of O&M costs, improved demonstration of capa bility , and integration into existing State certification programs . Inflation has increased all operation and maintenance and labor costs accordingl y. The increase in the respondent universe has increased the overall burden costs for the respondents. Stat es integrating this oversight into an existing certification program were not included in the previous burden estimate. As the States implement their certification programs, estimates will be adjusted
. **6(g) Burden Statement**  The annual reporting and recordkeeping burden for this collection is estimated to average 8 4.2 hours annuall y per respondent ( laboratory or State) (the combined total hours per ye ar for laboratories seeking certification and States integrating Cryptosp oridium certification programs divided by

65 total respondents ). Burden means the total time, effort, or financial resources expended b y persons to generate, maintain, retain, or disclose or provide information to or for a Federal agency. This

### 15

includes the time needed to review instructions; develop, acquire, install, and utilize technology and sy stems for the purposes of collecting, validating, and verifying information, processing and maintaining information, and disclosing and providing information; ad just the existing way s to comply with any previously

applicable instructions and requirements; train personnel to be able to respond to a collection of information; search data sources; complete and review the collection of information; and transmit or oth erwise disclose the information. An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it display s a currently

valid OMB control number. The OMB control numbers for EPA’s regulations are listed in 40 CFR Part 9 (http://ecfr.gpoaccess.gov/cgi/t/text/text
idx?c=ecfr&tpl=/ecfrbrowse/Title40/40cfr92_main_02.tpl ) and 48 CFR Chapter 15 (http://www.access.gpo.gov/nara/cfr/waisidx_00/48cfrv6_00.html ). To comment on the Agency's need for this information, the accuracy of the provided burden estimates, and any sugges ted methods for minimizing respondent burden, including use of automated collection techniques , EPA has established a public docket for this I CR under Docket ID Number EPA
- HQ
- OW
- 2002
- 0011 which is available for online viewing at www.regulations.gov, or in person viewing at the Water Docket in the EPA Docket Center (EPA/DC), EPA West, Room 3334, 1301 Constitution Avenue, NW, Washington, D.C. The EPA Docket Center Public Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday through Friday , excluding legal holiday s. The telephone number for the Reading Room is (202) 566
- 1744, and the telephone number for the W ater Docket is (202) 566
- 2426. An electronic version of the public docket is available at www.regulations.gov. This site can be used to submit or view public comments, access the index listing of the contents of the public docket, and to access those documents in the public docket that are available electronicall y. When in the system, select “search,” then key

in the Docket ID Number identified above. Al so, you can send comments to the Office of Information and Regulatory Affairs, Office of Management and Budget, 725 17th Street, NW, Washington, D
. C
. 20503, Attention: Desk Officer for EPA. Please include the EPA Docket ID Number EPA
- HQ
- OW
- 2002
- 0011 and OM B Control Number 2040
- 0246 in any correspondence.

### A
- 1 

# APPENDIX 

# A Federal Register Notice : Laboratory

Quality Assurance Evaluation Program/ In formation Collection Request [Published December 29, 2000]

### A
- 2 **ENVIRONMENTAL PROTECTION AGENCY**

[FRL
- 7152
- 6] **Laboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium**  **under the Safe Drinking Water Act; Agency Information Collection: Proposed Collection;**  **Comment Request AGENCY:**  Environmental Protection Agency
. **ACTION** : Notice; Request for Comment.

---------------------------------------------------------------------------- **SUMMARY:** Today ’s notice invites comment on the U.S. Environmental Protection Agency ’s (EPA’s) proposed Labor atory Quality Assurance Evaluation Program for Analy sis of Cryptosporidium under the Safe Drinking Water Act (Lab QA Program) (Section I). EPA also plans to submit to the Office of Management and Budget (OMB) for review and approval an Information Collection Request (ICR) associated with information collections under the proposed Lab QA Program (Section II). EPA is requesting comments on specific aspects of the proposed Lab QA Program and the ICR. Finally , EPA solicits comments on its intention to seek an emergency clearance from OMB to begin collecting data from laboratories that are interested in participating in the Lab QA Program prior to OMB’s final approval of the ICR. **DATES:** The Agency

requests comments on today ’s notice. Comments must be received or post - marked b y midnight May 3, 2002. If EPA does not receive adverse comments on or before April 3, 2002 regarding EPA’s request for an emergency clearance, the Agency intends t o seek a 90
- day

emergency

clearance from OMB to begin collecting data from laboratories that are interested in participating in the Lab QA Program. **ADDRESSES:** Please send an original and three copies of your written comments and enclosures (including r eferences) to the W
- 01
- 17 Comment Clerk, Water Docket (MC
4101), EPA, 1200 Pennsylvania Avenue, NW, Washington, DC 20460. Due to the uncertainty of mail delivery in the Washington, DC area, in order to ensure that all comments are received please send a separate copy of your comments via electronic mail (e
- mail) to Mary

Ann Feige, EPA, Office of Ground Water and Drinking Water, feige.maryann@epa.gov , or mail to the attention of Mary Ann Feige, EPA, Technical Support Center, 26 West Martin Luther King Dri ve (MS
- 140), Cincinnati, Ohio 45268. Hand deliveries should be delivered to: EPA’s Water Docket at 401 M Street, SW, Room EB57, Washington, DC 20460. Please make certain to reference EPA ICR No. 2052.02 and OMB Control No. 2040
- 0229. **FOR FURTHER INFORMATION:** For a cop y of the I CR, contact Sharon Gonder at EPA by phone at (202) 564
- 5256 or by email at gonder.sharon@epa.gov or download off the Internet at http://www.epa.gov/icr and refer to EPA I CR No. 2052.02. For technical inquiries, contact Mary

Ann Feige, EPA, Office of Ground Water and Drinking Water, Technical Support Center, 26 West Martin Luther King Drive (MS
- 140), Cincinnati, Ohio 45268, fax number, (513) 569
- 7191, e
- mail address, feige.maryann@epa.gov. **SUPPLEMENTARY INFORMAT ION:** 

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- 3

**Submission of comments.** Individuals who want EPA to acknowledge receipt of their comments should enclose a self
- addressed, stamped envelope. No facsimiles (faxes) will be accepted. Comments may also be submitted electronicall y to ow - docket@epamail.epa.gov
. Electronic comments must be submitted as an ASCII, WP5.1, WP6.1 or WP8 file avoiding the use of special characters and form of encry ption. Electronic comments must be identified by docket number W
- 01
- 17. Comments and data will also be accepte d on disks in WP5.1, 6.1, 8 or ASCII file format. Electronic comments on this notice may be filed online at many

Federal Depository

L ibraries. **Availability of docket.** The record for this notice has been established under docket number W
- 01
- 17, and inclu des supporting documentation as well as printed, paper versions of electronic comments. The record is available for inspection from 9 a.m. to 4 p.m., Monday through Friday , excluding legal holiday s at the Water Docket, EB 57, EPA Waterside Mall, 401 M Street, SW, Washington, DC 20460. For access to docket materials, please call (202) 260
- 3027 to schedule an appointment. **Section I: Laboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium**  **under the Safe Drinking Water Act** In September 2000, the Stage 2 Microbial and Disinfection By products Federal Advisory Committee (Committee) signed an Agreement in Principle (Agreement) (65 FR 83015, Dec. 29, 2000) (EPA, 2000) with consensus recommendations for two future drinking water regulatio ns: the Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) and the Stage 2 Disinfectants and Disinfection By products Rule. The LT2ESWTR is to address risk from microbial pathogens, specifically Cryptosporidium , and the Stage 2 DBPR is to address risk from disinfection by products. The Committee recommended that the LT2ESWTR require public water s ys tems (PWSs) to monitor their source water for Cryptosporidium using EPA Method 1622 or EPA Method 1623. Additional Cryptosporidium treatment requirements for PWSs would be based on the source water Cryptosporidium levels. EPA intends to take into account the Committee’s advice and recommendations embodied in the Agreement when developing the regulations. To support Cryptosporidium monitoring under the LT2ESWTR, the Committee Agreement recommended that “compliance schedules for the LT2ESWTR...be tied to the availability

of sufficient anal ytical capacity at approved laboratories for all large and medium
- size affected s ys tems to initiate Cryptosporidium and E.coli monitoring...”(65 FR 83015, Dec. 29, 2000) (EPA, 2000). Further, the Agreement recommended that Cryptosporidium monitoring b y large and medium sy stems begin within six months following rule promulgation. Given the time necessary for EPA to approve a sufficient number of laboratories to assure adequate capacity for LT2ESWTR monitoring, EPA

### A
- 4

would need to begin laboratory evaluation prior to promulgation of the rule in order to accommodate such an implementation schedule. Another factor that warrants initiation of the Lab QA Program prior to promulgation of the LT2ESWTR is grandfathering of monitoring data. The Agreement recommends that s ys tems with “historical” Cryptosporidium data that are equivalent to data that would b e collected under the LT2ESWTR be afforded the opportunity to use those “historical” (grandfathered) data in lieu of collecting new data under LT2ESWTR. EPA intends to propose such grandfathering provisions in the LT2ESWTR. If EPA indicates that laborato ries meet the criteria in the Lab QA Program described today prior to finalizing the LT2ESWTR, systems could develop monitoring data prior to the LT2ESWTR in anticipation of using it as grandfathered data. EPA’s Office of Ground Water and Drinking Water p lans to request from OMB an emergency

clearance that would enable expeditious implementation of a voluntary Lab QA Program to support Cryptosporidium monitoring under the LT2ESWTR. As such, the Agency

could begin to evaluate laboratories that can reliably measure for Cryptosporidium using EPA Method 1622 and Method 1623. During the effective period of the emergency

clearance, EPA intends to submit to OMB for review and approval a final I CR in order to continue data collection for the Lab QA Program. As part of today ’s notice, EPA is inviting comment on the L ab QA Program. Under the Lab QA Program, EPA would evaluate labs on a case
- by
- case basis through evaluating their capacity and competency to reliably

measure for the occurrence of Cryptosporidium in surface water using EPA Method 1622 or EPA Method 1623. The intent of this notice is not to propose establishing the Lab QA Program through a rulemaking. Rather, the criteria described in section I .C. are intended to provide guidance to laboratories that are interested in participating in the Lab QA Program. EPA has not yet proposed rulemaking on use of such “historical” data nor on the methods themselves under the LT2ESWTR. As noted above, EPA intends to propose allowing s ys tems to use equivalent “historical” data in lieu of collecting new data. EPA anticipates the data generated b y labs which meet the evaluation criteria would be very high quality, thus increasing the likelihood that such data would warrant consideration as acceptable “grandfathered” data. However, lab evaluation would not guarantee that data generated will be acceptable as “grandfathered” data, nor would failure to meet evaluation criteria necessarily preclude use of “grandfathered” data. For these reasons, EPA is not establishing th e Lab QA Program through rulemaking, but rather as a discretionary

and voluntary

program under the Safe Drinking Water Act, section 1442 (42 USC 300j
- 1(a)). **A. What is the purpose of the laboratory quality assurance evaluation program?** The purpose of the Lab QA Program is to identify laboratories that can reliably measure for the occurrence of Cryptosporidium in surface water. Existing laboratory certification programs do not include Cryptosporidium anal ys is. This program is designed to assess and confi rm the capability of laboratories to perform Cryptosporidium anal ys es. The program will assess whether laboratories meet the recommended personnel and laboratory

criteria in today ’s notice. This evaluation program is voluntary for laboratories. In the LT2ESWTR, however, EPA intends to require s ys tems to use

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- 5

approved (or certified) laboratories when conducting Cryptosporidium monitoring under the LT2ESWTR. **B. Why has EPA selected Methods 1622 and 1623 as the basis for determining the data quality of l** 

**aboratories that measure for Cryptosporidium?** EPA Method 1622 and EPA Method 1623 were developed as improved alternatives to the ICR Protozoan Method (EPA, 1996). EPA validated Method 1622 for the determination of Cryptosporidium in ambient water in Augus t 1998 and distributed an interlaboratory

validated draft method in January 1999. In addition, EPA validated Method 1623 for the simultaneous determination of Cryptosporidium (and Giardia ) in ambient water in February 1999 and distributed a validated draf t method in April 1999. In April 2001, EPA revised and updated Method 1622 (EPA
- 821
- R
- 01
- 026) (EPA, 2001a) and Method 1623 (EPA
- 821
- R
- 01
- 025) (EPA, 2001b) based on the following: laboratory

feedback, the development of equivalent filters and antibodies fo r use with the methods, and method performance data generated during the ICR Supplemental Survey s (EPA, 2001e). The results of these studies are documented in the Method 1622 interlaboratory validation study

report (EPA
- 821
- R
- 01
- 027) (EPA, 2001c) and the M ethod 1623 interlaboratory validation study

report (EPA
- 821
- R
- 01
- 028) (EPA, 2001d). **C. What criteria should I use to determine if my laboratory should apply?** A laboratory

that is interested in participating in the Lab QA Program currently should be operating in accordance with its QA plan (developed b y the laboratory) for Cryptosporidium analy ses. In addition, an interested laboratory should demonstrate its capacity and competency to analyze Cryptosporidium using the following recommended criteria: **1. Recommended personnel criteria:**  Principal Analy st/Supervisor ( 1 per laboratory) should have:

· BS/BA in microbiology or closel y related field

· A minimum of 1 year of continuous bench experience with Cryptosporidium and immunofluorescent assay (IFA) microscop y

· A minimum of six months experience using EPA Method 1622 and/or EPA Method 1623

· A minimum of 100 samples anal yz ed using EPA Method 1622 and/or EPA Method 1623 (minimum 50 samples if the person was an analy st approved to conduct analy sis for the ICR Protozoan Method (EPA, 1996)) for the specific anal ytical procedure they will be using

· Submit to EPA, along with the application package, resumes detailing the qualifications of the laboratory ’s proposed principal analy st/supervisor Other Anal ys ts (no minimum number of anal ys ts per laboratory) should have:

· Two years of college (or equivalent) in microbiology or closel y related field

· A minimum of six months of continuous bench experience with Cryptosporidium and IFA microscopy

### A
- 6

· A minimum of three months experience using EPA Method 1622 and/or EPA Method 1623

· A minimum of 50 samples anal yz ed using EPA Method 1622 and/or EPA Method 1623 (minimum 25 samples if the person was an analy st approved to conduct analy sis for th e ICR Protozoan Method) for the specific analy tical procedures they

will be using

· Submit to EPA, along with the application package, resumes detailing the qualifications of the laboratory ’s proposed other analy sts Technician(s) (no minimum number of technicians per laboratory) should have:

· Three months experience with the specific parts of the procedure they will be performing

· A minimum of 50 samples anal yz ed using EPA Method 1622 and/or EPA Method 1623 (minimum 25 samples if the person was an analy st approved to conduct analy sis for the ICR Protozoan Method) for the specific analy tical procedures they

will be using

· Submit to EPA, along with the application package, resumes detailing the qualifications of the laboratory ’s proposed technician(s) **2. Recommended laboratory criteria:** 

· Appropriate instrumentation as described in EPA Methods 1622 and 1623 (EPA, 2001a,b)

· Equipment and supplies as described in EPA Methods 1622 and 1623 (EPA 2001a, 2001b)

· Detailed laboratory standard operating procedures fo r each version of the method that the laboratory

will use to conduct the Cryptosporidium anal ys es

· Laboratory

should provide a current cop y of the table of contents of their laboratory ’s quality assurance plan for protozoa anal ys es

· EPA Method 1622 or EPA M ethod 1623 initial demonstration of capability (IDC) data, which include precision and recovery (IPR) test results and matrix spike/matrix spike duplicate (MS/MSD) test results for Cryptosporidium . EPA intends to evaluate the IPR and MS/MSD results agains t the performance acceptance criteria in the April 2001 version of EPA Method 1622 or EPA Method 1623 (EPA, 2001a, 2001b). **D. How can I obtain an application package?** After the OMB clearance described above, EPA plans to make applications available on EPA ’s website at www.epa.gov/safewater/cry ptolabapproval.html . Completed applications should be sent to: EPA’s L aboratory Quality Assurance Evaluation Program Coordinator, c/o Dy ncorp I&ET, Inc., 6101 Stevenson Avenue, Alexandria, VA 22304
- 3540. If a labor atory does not have access to the Internet, the laboratory may contact Dy

ncorp I&ET, Inc. to request an application package. **E. If I demonstrate my laboratory’s capacity and competency according to the the personnel and laboratory criteria, what do I d**  **o next?** After the laboratory

submits to EPA an application package including supporting documentation, EPA intends to conduct the following steps to complete the process: 1. Upon receipt of a complete package, EPA contacts the laboratory for follow
- up i nformation and to schedule participation in the performance testing program. 2. EPA sends initial proficiency testing (IPT) samples to the laboratory (unless the laboratory has already successfully anal yz ed such samples under EPA‘s Protozoan PE program). IPT

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- 7

samples packets consist of eight spiked samples shipped to the laboratory within a standard matrix. 3. The laboratory

anal yz es IPT samples and submits data to EPA. 4. EPA conducts an on
- site evaluation and data audit. 5. The laboratory

anal yz es ongoing proficiency testing ( OPT ) samples three times per year and submits the data to EPA. OPT sample packets consist of three spiked samples shipped to the laboratory

within a standard matrix. 6. EPA contacts laboratories by letter within 60 day s of their laboratory on - site evaluation to confirm whether the laboratory has demonstrated its capacity and competency for participation in the program. **F. My laboratory has already submitted initial demonstration of capability (IDC) and initial performance**  **testing (IPT) data as part of the EPA Protozoan Performance Evaluation (PE) Program. Do I have to perform this demonstration testing again?**  No. If a laboratory

currently participates in the EPA Protozoan PE Program and acceptable IDC and IPT data have already been submitted (for the version of the method that the laboratory

will use to conduct Cryptosporidium analy ses), EPA would not expect the laboratory to repeat IDC and IPT analy ses. **Section II: Paperwork Reduction Act** The information collection requirements in this notice have been submitted for approval to the OMB under the Paperwork Reduction Act , 44 U.S.C. 3501 et seq . An I CR document has been prepared b y EPA (ICR No. 2052.02) and a copy may be obtained from Susan Auby b y mail at Collection Strat egies Division; EPA (2822); 1200 Pennsylvania Ave., NW, Washington, DC 20460, by email at auby .susan@epamail.epa.gov, or by calling (202) 260
- 4901. A copy may also be downloaded off the internet at http://www.epa.gov/icr. Since the EPA would solicit info rmation in application packages, including supporting documentation, anal ytical data, and other pertinent information from laboratories that are interested in participating in the voluntary L ab QA Program, the Agency is required to submit an ICR to OMB for review and approval. Entities potentially affected by

this action include public and private laboratories that wish to be evaluated to determine if they can reliably

measure for the occurrence of Cryptosporidium in surface waters that are used for drinki ng water sources using EPA Method 1622 or Method 1623. The burden estimate for the Lab QA Program information collection includes all the burden hours and costs required for gathering information, and developing and maintaining records associated with the Lab QA Program. The annual public reporting and recordkeeping burden for this collection of information is estimated for a total of 60 respondents and an average 78 hours per response for a total of 4,676 hours at a cost of $123,650. This estimate assum es that laboratories participating in the L ab QA program have the necessary equipment needed to conduct the analy ses. Therefore, there are no start
- up costs. The estimated total annual capital costs is $0.00. The estimated Operation and Maintenance (O&M) costs is $133,880. Burden means the total time, effort, or financial resources expended b y persons to generate, maintain, retain, or disclose or provide information to or for a Federal agency. This includes the time needed to review instructions; develop , acquire, install, and utilize technology and sy stems for the purposes of collecting, validating, and verifying information, processing and maintaining information, and disclosing and providing information; adjust the existing way s to comply with any prev iously

applicable instructions and requirements; train personnel to be able to respond to a collection of information; search data sources; complete and review the collection

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- 8

of information; and transmit or otherwise disclose the information. An Agency

may not conduct or sponsor, and a person is not required to respond to a collection of information unless it displays a currently valid OMB control number. The OMB control numbers for EPA's regulations are listed in 40 CFR Part 9 and 48 CFR Chapter 15. Comm ents are requested on the Agency's need for this information, the accuracy of the provided burden estimates, and any suggested methods for minimizing respondent burden, including through the use of automated collection techniques. Send comments on the ICR to the Director, Collection Strategies Division; EPA (2822); 1200 Pennsylvania Ave., NW, Washington, DC 20460; and to the Office of Information and Regulatory Affairs, Office of Management and Budget, 725 17th St., N.W., Washington, DC 20503, marked "Attention: Desk Officer for EPA." Include the ICR number in any correspondence. Because OMB is required to make a decision concerning the ICR between 30 and 60 day s after March 4, 2002, a comment to OMB is best assured of having its full effect if OMB rece ives it by April 3, 2002. The final ICR approval notice will respond to any OMB or public comments on the information collection requirements contained in today ’s notice. **References** EPA. 1996. I CR Microbial L aboratory Manual. Office of Research and D evelopment. EPA/600/R
- 95/178. April 1996. EPA. 2000. Stage 2 Microbial and Disinfection By products Federal Advisory Committee Agreement in Principle. Federal Register. Vol. 65, pp. 83015
- 83024. December 29, 2000. EPA. 2001a. EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. Office of Water. Washington, DC 20460. EPA
- 821
- R
- 01
- 026. April 2001. EPA. 2001b. EPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. Office of Water. Washington, DC 2046 0. EPA
- 821
- R
- 01
- 025. April 2001. EPA. 2001c . Interlaboratory Validation Study

Results for Cryptosporidium Precision and Recovery for EPA Method 1622. Office of Water. Washington, DC 20460. EPA
- 821
- R
- 01
- 027. April 2001. EPA. 2001d. Interlaboratory Validation Study

Results for the Determination of Cryptosporidium and Giardia Using EPA Method 1623. Office of Water. Washington, DC 20460. EPA
- 821
- R
- 01
- 028. April 2001. EPA. 2001e. Implementation and Results of the Information Collection Rule Supplemental Survey s. Office of Water. Washington, DC 20460. EPA
- 815
- R
- 01
- 003. February 2001. ________________________Date

### B
- 1 

# APPENDIX 

# B Federal Register Notice : Laboratory

Quality Assurance Evaluation Program/ In formation Collection Request [Published March 4, 2002]

### B
- 2 **ENVIRONMENTAL PROTECTION AGENCY**

**[FRL -**  **] Agency Information Collection Activities: Submission for OMB**  **Review; Comment Request;**  EPA Laboratory Quality Assurance Evaluation Program for Analy sis of Cryptosporidium under the Safe Drinking Water Act/ Laboratory approval for the Long Term 2 Enhanced Surface Water Treatment Rule **AGENCY** : Environmental Protection Agency (EPA). **ACTION** : Notice. **SUMMARY** : In compliance with the Paperwork Reduction Act (44 U.S.C. 3501 et seq .), this document announces that the following Information Collection Request (I CR) has been forwarded to the Office of Management and Budg et (OMB) for review and approval: EPA Laboratory

Quality Assurance Evaluation Program for Anal ys is of Cryptosporidium under the Safe Drinking Water Act, OMB Control Number 2040
- 0246, expiration date of July 31, 2002. The I CR describes the nature of the in formation collection and its expected burden and cost; where appropriate, it includes the actual data collection instrument. **DATES** : Comments must be submitted on or before [Insert date 30 day s after publication in the FEDERAL REGISTER]. **ADDRESSES** : Send comments, referencing EPA I CR No.2067.02 and OMB Control No.2040
- 0246, to the following addresses: Susan Aub y, U.S. Environmental Protection Agency, Collection Strategies Division (Mail Code 2822), 1200 Pennsylvania Avenue, N.W., Washington, DC 20460; and to Office of Information and Regulatory Affairs, Office of Management and Budget (OMB), Attention: Desk Officer for EPA, 725 17 th 

### Street, N.W., Washington, DC 20503. **FOR FURTHER INFORMATION CONTACT** : For a cop y of the ICR contact Susan Aub y at EPA by phone at (202) 260
- 4901, by E
- mail at auby .susan@epamail.epa.gov, or download off the Internet at http://www.epa.gov/icr and refer to EPA I CR No. 2067.02, the I CR number has changed from the last notice. All requests should refer to EPA ICR No. 2067.02 and not EPA ICR No. 2052.02. For technical inquiries, contact Mary Ann Feige, EPA, Office of Ground Water and Drinking Water, Technical Support Center, 26 West Martin Luther King Drive (MS
- 140), Cincinnati, Ohio 45268, fax number, (513) 569
- 7191, e
- mail address, feig e.maryann@epa.gov. **SUPPLEMENTARY INFORMATION** : **Title** : EPA L aboratory

Quality Assurance Evaluation Program for Anal ys is of Cryptosporidium under the Safe Drinking Water Act (OMB Control No. 2040
- 0246 ; EPA ICR No. 2067.01 ) expiring 7/31/02 . This is a reque st for extension of a currentl y approved collection. Abstract: Section I: Laboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium Under the Safe Drinking Water Act In September 2000, the Stage 2 Microbial and Disinfection By produc ts Federal Advisory Committee (Committee) signed an Agreement in Principle (Agreement) (65 FR 83015, Dec. 29, 2000) (EPA, 2000) with consensus recommendations for two future drinking water regulations: The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) and the Stage 2 Disinfectants and Disinfection By products Rule. The LT2ESWTR is to address risk from microbial pathogens, specificall y Cryptosporidium , and the Stage 2 DBPR is to address risk from disinfection by

products. The Committee recommend ed that the LT2ESWTR require public water sy stems (PWSs) to monitor their source water for Cryptosporidium using EPA Method 1622 or

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EPA Method 1623. Additional Cryptosporidium treatment requirements for PWSs would be based on the source water Cryptosporidi um levels. To support Cry ptosporidium monitoring under the LT2ESWTR, the Committee Agreement recommended that ``compliance schedules for the LT2ESWTR * * * be tied to the availability of sufficient anal ytical capacity at approved laboratories for all large and medium
- size affected sy stems to initiate Cryptosporidium and E.coli monitoring * * * '' (65 FR 83015, Dec. 29, 2000) (EPA, 2000). Further, the Agreement recommended that Cryptosporidium monitoring by large and medium s ys tems begin within six months following rule promulgation. Given the time necessary for EPA to approve a sufficient number of laboratories to assure adequate capacity for LT2ESWTR monitoring, EPA would need to begin laboratory

evaluation prior to promulgation of the rule in order to accommodate such an implementation schedule. Another factor that warrants initiation of the Lab QA Program prior to promulgation of the LT2ESWTR is grandfathering of monitoring data. The Agreement recommends that s ys tems with ``historical'' Cryptosporidium data that are equivalent to data that would be collected under the LT2ESWTR be afforded the opportunity to use those ``historical'' (grandfathered) data in lieu of collecting new data under LT2 ESWTR. EPA intends to propose such grandfathering provisions in the LT2ESWTR. If EPA indicates that laboratories meet the criteria in the Lab QA Program described today prior to finalizing the LT2ESWTR, systems could develop monitoring data prior to the LT 2ESWTR in anticipation of using it as grandfathered data. Under the Lab QA Program, EPA would evaluate labs’ capacity and competency

to reliably

measure for the occurrence of Cryptosporidium in surface water using EPA Method 1622 or EPA Method 1623. The intent of this notice is not to propose establishing the Lab QA Program through a rulemaking. Rather, the criteria described in section I.C. are intended to provide guidance to laboratories that are interested in participating in the Lab QA Program. EPA anti cipates the data generated b y l abs which meet the evaluation criteria would be very high quality , thus increasing the likelihood that such data would warrant consideration as acceptable ``grandfathered'' data. However, lab evaluation would not guarantee th at data generated will be acceptable as ``grandfathered'' data, nor would failure to meet evaluation criteria necessarily

preclude use of ``grandfathered'' data. For these reasons, EPA is not establishing the Lab QA Program through rulemaking, but rather a s a discretionary and voluntary program under the Safe Drinking Water Act, section 1442 (42 USC 300j
- 1(a)). **A. What Is the Purpose of the Laboratory Quality Assurance Evaluation Program?** The purpose of the Lab QA Program is to identify laboratories that ca n reliably measure for the occurrence of Cryptosporidium in surface water. Existing laboratory certification programs do not include Cry ptosporidium anal ys is. This program is designed to assess and confirm the capability

of laboratories to perform Cry ptosp oridium anal ys es. The program will assess whether laboratories meet the recommended personnel and laboratory criteria in today's notice. This evaluation program is voluntary for laboratories. In the LT2ESWTR, however, EPA intends to require s ys tems to use approved (or certified) laboratories when conducting Cryptosporidium monitoring under the LT2ESWTR. **B. How Can I Obtain an Application Package?** After the OMB clearance described above, EPA plans to make applications available on EPA's website at www.epa.gov/safewater/ cry ptolabapproval.html. Completed applications should be sent to: EPA's Laboratory Quality Assurance Evaluation Program Coordinator, c/o Dy nCorp, 6101 Stevenson Avenue, Alexandria, VA 22304
- 3540. If a laboratory does not have access to th e Internet, the laboratory may contact Dy

nCorp to request an application package. Applications may

be submitted at any

time.

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**C. If I Demonstrate My Laboratory's Capacity and Competency According to the Personnel and Laboratory Criteria, What Happens Next?**  After the laboratory

submits to EPA an application package including supporting documentation, EPA intends to conduct the following steps to complete the process: 1) Upon receipt of a complete package, EPA contacts the laboratory for follow
- up information and to schedule participation in the performance testing program. 2) EPA sends initial proficiency testing (IPT) samples to the laboratory . IPT samples packets consist of eight spiked samples shipped to the laboratory within a standard matrix. 3) The laboratory

analyzes the IPT samples and submits data to EPA. EPA intends to have the laboratory ’s IPT data meet the IPT criteria of greater than 10% mean recovery and less than 71% relative standard deviation (these criteria were developed based on results from the first six rounds of the EPA PE program). This approach will be used unless unforeseen circumstances merit a reassessment of the approach. 4) EPA conducts an on
- site evaluation and data audit. Checklist for evaluation and audit is included in I CR. 5) The laboratory

analyzes ongoing proficiency testing (OPT) samples three times per year and submits the data to EPA. OPT sample packets consist of three spiked samples shipped to the laboratory

within a standard matrix. The results of the labo ratory ’s OPT data must meet the OPT criteria which will be calculated for each round of OPT testing using onl y the data from that round. EPA intends to calculate the lower limit as less than 2 standard deviations from the pooled mean using log it transfor med data and intends to calculate the maximum RSD as 2 times the pooled RSD. This approach will be used unless unforeseen circumstances merit a reassessment of the approach. 6) EPA contacts laboratories by letter within 60 day s of their laboratory on - site evaluation to confirm whether the laboratory has demonstrated its capacity and competency for participation in the program. An agency

may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB control number. The OMB control numbers for EPA's regulations are listed in 40 CFR part 9 and 48 CFR Chapter 15. The Federal Register document required under 5 CFR 1320.8(d), soliciting comments on this collection of information was publ ished on March 4, 2002 ( FR ). Three comments were received. Comments requested further information on the details of the Lab Quality Assurance Program. In response, EPA has added supplementary information to the ICR, including the program application, which includes the self
- audit checklist detailing the items that will be evaluated during the on
- site evaluation. EPA also has also developed a webpage to provide further information on the program. The website can be accessed at http://www.epa.gov/safewater/lt2/cla_final.html. Commenters expressed concern that the Lab QA Program does not address the Agency ’s obligation under the FACA Agreement in Principle to identify adequate laboratory

capacity to implement LT2ESWTR. The Lab QA Program does assess laboratory capacity through questions on the application on current and potential laboratory capacity to anal yz e Cryptosporidium samples and the on site evaluations. This information will be compiled as laboratory applications are received, and will be updated d uring on
- site evaluations. The on
- site evaluation will allow EPA to validate lab capacity reported to EPA.

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Comments were received on the burden estimates. Because laboratories that wish to begin using EPA Methods 1622 and 1623 are required by the methods to purchase the equipment necessary to demonstrate initial acceptable performance, and because this is a method requirement, rather than a program requirement (laboratories can perform the methods without ever participating in the program), the burden esti mates assume that no capital costs will be incurred b y laboratories participating in the program over and above the costs that would be incurred simply

to use the method. Because the program application requires the laboratories applying for approval under the program to submit initial performance data, laboratories that meet these requirements should already have the capacity to perform Methods 1622 or 1623 and therefore will not incur start
- up costs. Commenters wanted to know if training would be availabl e for labs needing help. EPA will provide limited training to laboratories needing assistance with the performance of Methods 1622 and 1623. Information on training will be posted on EPA’s website as it becomes available. Commenters wanted to know the earliest date that acceptable grandfathered data could be generated. EPA is aware of the issues regarding grandfathered data acceptability and will address these issues in the proposed LT2ESWTR. These issues are outside of the scope of this I CR. Burden St atement: The annual public reporting and record keeping burden for this collection of information is estimated to average 18 hours per response. Burden means the total time, effort, or financial resources expended b y persons to generate, maintain, retain, or disclose or provide information to or for a Federal agency . This includes the time needed to review instructions; develop, acquire, install, and utilize technology and sy stems for the purposes of collecting, validating, and verifying information, proces sing and maintaining information, and disclosing and providing information; adjust the existing ways to comply with any previously

applicable instructions and requirements; train personnel to be able to respond to a collection of information; search data s ources; complete and review the collection of information; and transmit or otherwise disclose the information. Respondents/Affected Entities: Testing Laboratories Estimated Number of Respondents: 60. Frequency of Response: 3 times per year. Estimated Total Annual Hour Burden: 4347 hours. Estimated Total Annualized Capital, O&M Cost Burden: $123,380. Send comments on the Agency's need for this information, the accuracy of the provided burden estimates, and any suggested methods for minimizing respondent burd en, including through the use of automated collection techniques to the addresses listed above. Please refer to EPA I CR No. 2067.02 and OMB Control No. 2040
- 0246 in any correspondence.

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- 1 

# APPENDIX 

# C Federal Register Notice : Laboratory

Quality Assurance Evaluation Program/ In formation Collection Request [Published October 26, 2005]

### C
- 2 ENVIRONMENTAL PROTECTION AGENCY [OW

–2002

–0011, FRL

– 7988

–7] **Agency Information Collection Activities; Submission to OMB for**  **Review and Approval; Comment Request**  ; **Laboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium Under the Safe Drinking Water Act, EPA ICR Number 2067.03, OMB**  **Control Number 2040**

**– 0246**  **AGENCY:** Environmental Protection Agency
. **ACTION:** Notice. **SUMMARY:** In compliance with the Paperwork Reduction Act (44 U.S.C. 3501 et seq.), this document announces that an Information Collection Request (ICR) has been forwarded to the Office of Management and Budget (OMB) for review and approval. This is a request to renew an existing approved collection. This I CR is scheduled to expire on October 31, 2005. Under OMB regulations, the Agency

may continue to conduct or sponsor the collection of information while this submission is pending at OMB. This ICR d escribes the nature of the information collection and its estimated burden and cost. **DATES:** Additional comments may be submitted on or before November 25, 2005. **ADDRESSES:** Submit your comments, referencing docket ID number OW

– 2002

–0012, to (1) EPA online using EDOCKET (our preferred method), by email to ow
- docket@epamail.epa.gov, or by mail to: EPA Docket Center, Environmental Protection Agency , Water Docket, Mail Code 4101T, 1200 Pennsylvania Ave., NW., Washington, DC 20460. **FOR FURTHER INFORMATION CONTA CT:**  Sean Conley , Office of Groundwater and Drinking Water, (Mail Code 4607M), Environmental Protection Agency , 1200 Pennsylvania Ave., NW., Washington, DC 20460; telephone number: 202

– 564

– 1781; fax number: 202

– 564

– 3767; e
- mail address: conley .sean@epa.gov . For technical inquiries, contact Carrie Moulton, EPA, Office of Ground Water and Drinking Water, Technical Support Center, 26 West Martin Luther King Drive (MS

– 140), Cincinnati, Ohio 45268; fax number: (513) 569

–7191; email address: moulton.carrie@epa.go v. **SUPPLEMENTARY INFORMATION:** EPA has submitted the following ICR to OMB for review and approval according to the procedures prescribed in 5 CFR 1320.12. On June 3, 2005 (70 FR 32607), EPA sought comments on this I CR pursuant to 5 CFR 1320.8(d). EPA has addressed the comments received. EPA has established a public docket for this ICR under Docket ID No. OW

– 2002

–0012, which is available for public viewing at the Water Docket in the EPA Docket Center (EPA/DC), EPA West, Room B102, 1301 Constitution Ave., NW., Washington, DC. The EPA Docket Center Public Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday

through Friday , excluding legal holiday s. The telephone number for the Reading Room is (202) 566

– 1744, and the telephone number for the Water Docket i s (202) 566

– 2426. An electronic version of the public docket is available through EPA Dockets (EDOCKET) at http://www.epa.gov/edocket. Use EDOCKET to submit or view public comments, access the index listing of the contents of the public docket, and to acce ss those documents in the public docket that are available electronically . Once in the sy stem, select ‘‘search,’’ then key in the docket ID number identified above. Any comments related to this I CR should be submitted to EPA and OMB within 30 day s of thi s notice. EPA’s policy is that public comments, whether submitted electronicall y or in paper, will be made available for public viewing in EDOCKET as EPA receives them and without change, unless the comment contains cop yrighted material, CBI, or other info rmation whose public disclosure is restricted b y statute. When EPA identifies a comment containing

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- 3

copy righted material, EPA will provide a reference to that material in the version of the comment that is placed in EDOCKET. The entire printed comment, including the copyrighted material, will be available in the public docket. Although identified as an item in the official docket, information claimed as CBI, or whose disclosure is otherwise restricted b y statute, is not included in the official public docket , and will not be available for public viewing in EDOCKET. For further information about the electronic docket, see EPA’s Federal Register notice describing the electronic docket at 67 FR 38102 (May 31, 2002), or go to http://www.epa.gov/edocket. Title: Laboratory Quality Assurance Evaluation Program for Anal ys is of Cry ptosporidium under the Safe Drinking Water Act. Abstract: Under the Laboratory Quality Assurance Evaluation Program, EPA evaluates labs on a case
- by
- case basis through evaluating their ca pacity and competency to reliabl y measure for the occurrence of Cry ptosporidium in surface water using EPA Method 1622 or EPA Method 1623. To obtain approval under the program, the laboratory must submit an application package and provide: a demonstration of availability of qualified personnel and appropriate instrumentation, equipment and supplies; a detailed laboratory standard operating procedure for each version of the method that the laboratory will use to conduct the Cry ptosporidium anal ys es; a current copy of the table of contents of their laboratory ’s quality assurance plan for protozoa anal ys es; and an initial demonstration of capability data for EPA Method 1622 or EPA Method 1623, which include precision and recovery test results and matrix spike/m atrix spike duplicate test results for Cry ptosporidium. After the laboratory submits to EPA an application package including supporting documentation, EPA and the laboratory conduct the following steps to complete the process: 1. EPA contacts the laborato ry for follow
- up information and to schedule participation in the performance testing program. 2. EPA sends initial proficiency testing samples to the laboratory (unless the laboratory has already successfully anal yz ed such samples under EPA’s Protozoan P E program). These sample packets consist of eight spiked samples shipped to the laboratory within a standard matrix. 3. The laboratory

anal yz es initial proficiency testing samples and submits data to EPA. 4. EPA conducts an on
- site evaluation and data au dit. 5. The laboratory

anal yz es ongoing proficiency testing samples three times per year and submits the data to EPA. These sample packets consist of three spiked samples shipped to the laboratory

within a standard matrix. 6. EPA contacts laboratories by letter within 60 day s of their laboratory onsite evaluation to confirm whether the laboratory has demonstrated its capacity and competency for participation in the program. The procedure for obtaining an application package, the criteria for demonstrating capacity and competency , and other guidance to laboratories that are interested in participating in the program, are provided at http://www.epa.gov/ safewater/lt2/cla_final.html. An agency

may not conduct or sponsor, and a person is not required to resp ond to, a collection of information unless it displays a currently valid OMB control number. The OMB control numbers for EPA’s regulations in 40 CFR are listed in 40 CFR part 9 and are identified on the form and/or instrument, if applicable. Burden Statem ent: The annual public reporting and recordkeeping burden for this collection of information is estimated to average 19 hours per response. Burden means the total time, effort, or financial resources expended b y persons to generate, maintain, retain, or di sclose or provide information to or for a Federal agency . This includes the time needed to review instructions; develop, acquire, install, and utilize technology and s ys tems for the purposes of collecting, validating, and verifying information, processing and maintaining information, and disclosing and providing information; adjust the existing way s to compl y with any previously

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- 4

applicable instructions and requirements; train personnel to be able to respond to a collection of information; search data source s; complete and review the collection of information; and transmit or otherwise disclose the information. Respondents/Affected Entities: Public and private water testing laboratories. Estimated Number of Respondents: 22. Frequency of Response: Three times per year. Estimated Total Annual Hour Burden: 3,980. Estimated Total Annual Cost: $275,000, includes $109,000 annualized capital or O&M costs. Changes in the Estimates: There is a decrease of 367 hours in the total estimated burden currentl y identified in the OMB Inventory of Approved I CR Burdens. This decrease is just an adjustment to the estimate. Dated: October 18, 2005. Sara Hisel
- McCoy , Acting Director, Collection Strategies Division. [FR Doc. 05

–21370 Filed 10

–25

–05; 8:45 am] BILLING CODE 6560

– 50

–P

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- 1 

# APPENDIX 

# D Federal Register Notice : Laboratory

Quality Assurance Evaluation Program/ In formation Collection Request [ Published February

25, 2009 ]

### D
- 2

ENVIRONMENTAL PROTECTION AGENCY [EPA
- HQ
- OW
- 2002
- 0011; FRL
- 8776
- 6] Agency Information Collection Activities; Proposed Collection; Comment Request; L aboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium Under the Safe Drinking Water Act (Renewal); EPA ICR No. 2067.04, OMB Control No. 2040
- 0246 AGENCY: Environmental Protection Agency (EPA). ACTION: Notice. SUMMARY: In compliance with the Paperwork Reduction Act (PRA) (44 U.S.C. 3501 et seq.), this do cument announces that EPA is planning to submit a request to renew an existing approved Information Collection Request (I CR) to the Office of Management and Budget (OMB). This I CR is scheduled to expire on May 31, 2009. This notice describes the current ``Laboratory

Quality Assurance Evaluation Program for Anal ys is of Cryptosporidium under the Safe Drinking Water Act,'' hereafter referred to as the ``Lab QA Program,'' and requests comment on both the program and the renewed paperwork requirements. DA TES: Comments must be submitted on or before April 27, 2009. ADDRESSES: Submit your comments, identified by Docket ID No. EPA
- HQ
- OW
- 2002
- 0011, by one of the following methods: http://www.regulations.gov: Follow the on
- line instructions for submitting c omments. Mail: Water Docket, Environmental Protection Agency , Mailcode: 2822T, 1200 Pennsylvania Ave., NW., Washington, DC 20460. Hand Delivery : Monday

through Friday , excluding legal holiday s. The telephone number for the Reading Room is (202) 566
- 1744, and the telephone number for the Water Docket is (202) 566
- 2426. Such deliveries are only accepted during the Docket's normal hours of operation, and special arrangements should be made for deliveries of boxed information. Instructions: Direct your comments to Docket ID No. EPA
- HQ
- OW
- 2002
- 0011. EPA's policy is that all comments received will be included in the public docket without change and may be made available online at http://www.regulations.gov, including any personal information provided, unless the comment includes information claimed to be Confidential Business In formation (CBI) or other information whose disclosure is restricted b y statute. Do not submit information that you consider to be CBI or otherwise protected using http://www.regulations. gov or e - mail. Please contact EPA prior to submitting CBI. The http://www.regulations.gov Web site is an ``anonym ous access'' s ys tem, which means EPA will not know your identity or contact information unless you provide it in the body of your comment. If you send an e
- mail comment directly to EPA without going through http://www.regulations.gov your e - mail address will be automatically

captured and included as part of the comment that is placed in the public docket and made available on the Internet. If you submit an electronic comment, EPA recommends that yo u include your name and other contact information in the body of your comment and with any disk or CD
- ROM you submit. I f EPA cannot read your comment due to technical difficulties and cannot contact you for clarification, EPA may not be able to consider your comment. Electronic

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files should avoid the use of special characters, any form of encry

ption, and be free of any defects or viruses. For additional information about EPA's public docket visit the EPA Docket Center homepage at http://www.epa.gov/epahome/dockets.htm. FOR FURTHER INFORMATION CONTACT: Carrie Miller, EPA, Office of Ground Water and Drinking Water, Technical Support Center, 26 West Martin Luther King Drive (MS
- 140), Cincinnati, Ohio 45268; e - mail address: miller.carrie@epa.gov. SUPPLEMENTARY INFORMATION: How Can I Access the Docket and/or Submit Comments? EPA has established a public docket for this ICR under Docket ID No. EPA
- HQ
- OW
2002
- 0011, which is available for online viewing at http://www.regulations.gov, or in person viewing at the Water Docket in the EPA Docket Center (EPA/DC), EPA West, Room 3334, 1301 Constitution Ave., NW., Washington, DC. The EPA/DC Public Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday through Friday , excluding legal holiday s. The telephone number for the Reading Room is 202
- 566
- 1744, and the telephone number for the Water Docket is 202
- 566
- 2426. Use http://www.regulations.gov to obtain a cop y of the draft collection of information, submit or view public comments, access the index listing of the contents of the docket, and to access those documents in the public docket that are available elec tronically . Once in the s ys tem, select ``search,'' then key in the docket ID number identified in this document. What Information I s EPA Particularl y Interested in? Pursuant to section 3506(c)(2)(A) of the PRA, EPA specifically solicits comments and information to enable it to: (i) Evaluate whether the proposed collection of information is necessary for the proper performance of the functions of the Agency , including whether the information will have practical utility ; (ii) Evaluate the accuracy of the Agency's estimate of the burden of the proposed collection of information, including the validity of the methodology

and assumptions used; (iii) Enhance the quality , utility, and clarity of the information to be collected; and ( iv) Minimize the burden of the collection of information on those who are to respond, including through the use of appropriate automated electronic, mechanical, or other technological collection techniques or other forms of information technolo gy, e.g., permitting electronic submission of responses. In particular, EPA is requesting comments from very small businesses (those that employ less than 25) on examples of specific additional efforts that EPA could make to reduce the paperwork burden for very small businesses affected by this collection. EPA is also interested in any other comments regarding the improvements to the Lab QA Program described in this notice. What Should IConsider When IPrepare M y Comments for EPA? You may find the following sugges tions helpful for preparing yo ur comments: 1. Explain your views as clearl y as possible and provide specific examples.

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- 4

2. Describe any assumptions that you used. 3. Provide copies of any t echnical information and/or data you used that support your views
. 4. If you estimate potential burden or costs, explain how you arrived at the estimate that yo u provide. 5. Offer alternative way s to improve the collection activity
. 6. Make sure to submit your comments by the deadline identified under DATES. 7. To ensure proper receipt by EPA, be sure to identify

the docket ID number assigned to this action in the subject line on the first page of your response. You may also provide the name, date, and Federal Register citation. What Information Collection Activity or ICR Does This Apply to? Affected entities: Entities potentially affected by

this action are public and private water testing laboratories. EPA estimates that a total of 65 laboratories will seek to attain or maintain EPA recognition under the Lab QA P rogram. This estimate includes 63 laboratories seeking continued recognition under the Lab QA Program and 2 laboratories seeking initial recognition. Title: L aboratory

Quality Assurance Evaluation Program for Anal ys is of Cryptosporidium under the Safe Drinking Water Act (Renewal). ICR numbers: EPA ICR No. 2067.04, OMB Control No. 2040
- 0246. ICR status: This I CR is currentl y scheduled to expire on May 31, 2009. An Agency

may not conduct or sponsor, and a person is not required to respon d to, a collection of information, unless it displays a currently valid OMB control number. Approved OMB control numbers for EPA's regulations in title 40 of the CFR are listed in 40 CFR part 9 of the Federal Register and displayed either by

publication of the Federal Register or by other appropriate means, such as on the applicable collection instrument or form. Abstract: In September 2000, the Stage 2 Microbial and Disinfection By products Federal Advisory Committee (Committee) signed an Agreement in Prin ciple (Agreement) (65 FR 83015, December 29, 2000) (EPA, 2000) with consensus recommendations for two future drinking water regulations: the Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) and the Stage 2 Disinfectants and Disinfection By produ cts Rule. The LT2ESWTR was to address risk from microbial pathogens, specifically Cryptosporidium . The Committee recommended that the LT2ESWTR require public water s ys tems (PWSs) to monitor their source water for Cryptosporidium using EPA Method 1622 or EP A Method 1623. Additional Cryptosporidium treatment requirements for PWSs would be based on the source water Cryptosporidium levels. EPA took into account the Committee's advice and recommendations as it developed the LT2ESWTR, which was published on January 5, 2006. Under the LT2ESWTR, EPA requires public water s ys tems to use approved laboratories when conducting Cryptosporidium monitoring. In the preamble to the LT2ESWTR as well as several other notices, EPA has described the criteria for approval of lab oratories to anal yz e Cryptosporidium samples under the LT2ESWTR. See 71 FR 727 (January 5, 2006) and 67 FR 9731 (March 4, 2002). The Lab QA Program, as revised, is described in this notice. The purpose of the Lab QA Program is to identify laboratories that can reliabl y measure for the occurrence of Cryptosporidium in surface water and to ensure that approved laboratories maintain that capability . Other, State
- based laboratory oversight programs do not currently address approval of laboratories for the Cryp tosporidium analy sis required by the LT2ESWTR.

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- 5

Through today's notice, EPA is inviting comment on refinements to the information collected to support EPA's L ab QA Program. As of May 2007, EPA concluded that sufficient laboratory

capacity exists for the LT 2ESWTR. As a result, EPA has generally postponed evaluation of additional laboratories, including commercial, county, municipal and utility laboratories, until further notice. Subject to the availability of resources, EPA will consider evaluation of State and EPA Regional laboratories on a case
- by
- case basis, based on the role that States and EPA Regions play in the certification and approval programs for laboratories. The Lab QA Program is continuously being refined and updated as new information and techn ologies become available. The program will continue to evolve and EPA will continue to revise and update burden estimates, as needed, with any subsequent ICR. Approved laboratories will have demonstrated, and are to continue to demonstrate, proficient and reliable detection and enumeration of Cryptosporidium in surface water sources for public water s ys tems. They will have passed all elements in the Lab QA Program and continue to successfull y participate in all program activities. Approved laboratories are responsible for notifying EPA of losses of key personnel or essential equipment and changes in policies or procedures that directl y affect the validity of data or any other change affecting the capability of the laboratory

including change in location. Participating laboratories are to also demonstrate ongoing capability and method performance b y following all applicable method quality control (QC) procedures, anal yz ing ongoing proficiency testing (PT) samples (generall y three times per ye ar), submitting requested data to EPA, and participating in periodic re
evaluations. The Lab QA Program procedures have been updated to reflect that the minimum recovery for Cryptosporidium in ongoing precision and recovery (OPR) samples is no w 22 percent, updated from th e original 11 percent. This updated minimum recovery is based on an updated data set and should provide a better assessment of laboratory performance than the original value for the following reasons: (1) The data set is more current and is based on more s amples (a total of 333); (2) 52 more laboratories are included in the data set; (3) data were generated using the 2005 version of Method 1623, which is the required version for LT2ESWTR anal ys es; (4) data were generated using filters currently used to anal yz e LT2ESWTR samples rather than those filters used originally ; and (5) the number of oocy sts spiked into the samples was unknown to the laboratories. Calculations for the updated criteria are available in Docket ID No. EPA
- HQ
- OW
- 2002
- 0011. L aboratories are to now document a minimum of 22 percent recovery for OPR samples in an updated QC chart prior to anal ys is of LT2ESWTR samples at the frequency

required in section 9.7 of the method. The ongoing PT sample packets generall y consist of three spiked samples shipped to the laboratory

within a standard matrix. I f a laboratory submits poor PT results, EPA may recommend additional follow
- up action to demonstrate that the laboratory's performance remains acceptable. Additional actions may include submission of PT slides to EPA, repeat anal ys es, providing additional QC data, and investigation of proble ms with reagents and equipment. Repeated failure to demonstrate laboratory capability

and acceptable method performance may result in suspension or downgrading of app roval status as outlined later in this section. EPA may re - evaluate laboratories participating in the program to verify Cryptosporidium laboratory

quality assurance (QA) on both an ``as
- needed'' and periodic basis (generall y not exceeding once every

three years). In the case of a periodic assessment, EPA will generall y notify the laboratory

that they are due for re
- evaluation and request a package with documentation of personnel status, equipment maintenance, standard operating procedures, training records, and QC charts. After the package has been received, it will be evaluated for

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completeness. EPA generally contacts the laboratory

within 15 day s of package submission if information is missing. When a complete package has been received, the following steps will complete the process: 1. The laboratory

will send positive staining control and OPR slides for evaluation by EPA. 2. The labo ratory

will order blind slides spiked with Cryptosporidium from a qualified vendor for each anal ys t. Each analy st will perform an independent count of one slide. The results and slides will be submitted to a technical auditor. 3. EPA will schedule an on
- l ine Internet anal ys t verification of performance for microscopists to demonstrate their ability to identify Cryptosporidium oocysts. 4. EPA conducts a one
- day on - site evaluation that will primarily focus on method performance and data recording. Laboratory personnel will be asked to order blind oocy st suspensions for use in sample and IMS control spiking in the presence of an auditor, and then complete the anal ys es within applicable method holding times and send results to EPA. 5. EPA will send the laboratory a report detailing all findings, generally within 60 day s after the evaluation is complete. The laboratory is then asked to provide written responses to any deficiencies identified in the report within 60 day s. Provided all responses to the deficienci es cited in the report are acceptable, the Lab QA Program will then base its decision for continued laboratory

approval on PT results, quality of the positive control and OPR slide, slide counts, Internet anal ys t verification, on
- site evaluation and recove ry values for blind anal ys es initiated during the on
- site evaluation. State and EPA Regional Laboratories may contact the laboratory

approval manager regarding new application submissions. Subject to available resources, EPA estimates that up to two State or EPA Regional Laboratories will seek first
- time approval each year. Laboratories seeking approval under the program must submit an application package and provide: a demonstration of availability of qualified personnel and appropriate instrumentation, equipment and supplies; detailed laboratory standard operating procedures; a current copy of the table of contents of their laboratory's QA plan for protozoa anal ys es; and an initial demonstration of capability data for EPA Method 1623, which includes initial precision and recovery IPR test results and matrix spike/matrix spike duplicate (MS/MSD) test results for Cryptosporidium
. After EPA completes its review of the application, the Agency will contact the laboratory for follow
- up information and to schedule shipment of initial PT samples consisting of eight spiked samples within a standard matrix. EPA then generally conducts an on
- site evaluation and data audit. Further information is provided at http://www.epa.gov/safewater/disinfection/lt2/lab_home.html. The Agency notes that completion of an application b y a laboratory does not ensure that the Agency will act on the laboratory's request; interested laboratories are encouraged to contact the laboratory approval manager prior to investing substantial effor t towards their application. Further, a decision by the Agency

to review an application, to send initial PT samples, and/or to schedule or conduct an on
- site evaluation and data evaluation, does not ensure that the review process will be completed or that the laboratory

will ultimately

be approved. Decisions will be made based on the facts associated with a particular application and actions will be taken as Agency resources permit. Approved laboratories that do not continue to meet the criteria for the La b QA Program may have their status downgraded to provisional or have their approval suspended. Details of the basis for downgrading or suspending a laboratory's approval are provided in the section entitled ``Clarification of Basis and Procedures for Downgrading/Suspending Approval for Laboratories for the Anal ys is of Cryptosporidium in Water Under the Long Term 2 Enhanced Surface Water

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Treatment Rule'' (see the following section). Provided EPA has sufficient resources to review requests for upgrade or rein statement, laboratories may have to undertake additional activities such as anal yz ing additional PT samples, undergoing an on
- site evaluation, and/or counting blind spiked slides in order to have their status upgraded or their approval reinstated. Details regarding additional activities that may be required are provided in the next section. Clarification of Basis and Procedures for Downgrading/Suspending Approval of Laboratories for the Anal ys is of Cryptosporidium in Water Under the L on g Term 2 Enhanced Surface Water Treatment Rule EPA's Office of Ground Water and Drinking Water, in the Office of Water, has developed a detailed description of the procedures and criteria used in actions concerning approving, downgrading and suspending laboratories for anal ys is of drinking water contaminants. In order to assume primary enforcement responsibility for the drinking water regulations, a State must either have available laboratory facilities, approved by

the Administrator, capable of conducting anal ytical measurements of drinking water contaminants, or establish and maintain its own program for approval of laboratories. States wishing to adapt these procedures and criteria for their own approval program should revise it to accuratel y reflect their State approval program. This section is intended to clarify EPA's intended practices and procedures for laboratory approval, downgrading or suspension for analy sis of Cryptosporidium under the Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) and to reflect g ood laboratory practice and standard proficiency

evaluation in the industry; it is not a regulation. While EPA intends to generall y follow the procedures laid out in this section, not every situation is reflected in these procedures and EPA may need to address case - specific situations in way s that differ from the procedures spelled out here. EPA welcomes comment on these procedures and may decide to revise them at any time in the future to reflect changes to its approach or to clarify and update the text. ``Approved Laboratories'' have demonstrated, and continue to demonstrate, proficient and reliable detection and enumeration of Cryptosporidium in surface water sources for public water sy stems. They

have passed all elements in the Lab QA Program and continue to successfull y participate in all program activities. Approved Laboratories notify the Approval Authority

(EPA individual(s) administering the program or State individual(s) administering an equivalent laboratory

certification program) of loss of key p ersonnel or essential equipment, change in policies or procedures that directl y affect the validity of data, and any other change affecting the capability of the laboratory

including change in location. ``P rovisionally

Approved L aboratories'' have deficiencies but demonstrate their ability to consistently

produce data of known quality . They continue to successfull y participate in all Lab QA Program activities. A Provisionally Approved Laboratory

may anal y ze drinking water samples for LT2ESWTR compliance purposes if the laboratory has identified themselves as provisionally

approved to their clients and any reports clearl y state that the laboratory's status is ``provisionally

approved.'' ``Not Approved'' d esignates a laboratory that has either not participated in the Lab QA Program, or has applied to the program but possesses deficiencies and, in the opinion of the Approval Authority, does not consistently produce data that has met all applicable method QC requirements or has falsified data.

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Basis for Downgrading to ``Provisionally Approved'' Status An Approved Laboratory (referred to as ``laboratory'') may be downgraded to ``P rovisionally

Approved'' status for Cryptosporidium for any of the following rea sons: Failure to anal yz e samples for the LT2ESWTR according to the December 2005 version of EPA Method 1623 or EPA Method 1622, including all QA/QC criteria; Failure to document a minimum of 22 percent for on
- going precision and recovery values in an upd ated QC chart prior to anal ys is of LT2ESWTR samples at the frequency required in section 9.7 of the method; Failure to demonstrate proficiency based upon acceptable matrix spike recoveries for all modifications of the method procedures per Section 9.1.2 o f the method; Failure to submit valid Proficiency Test (PT) results or meet PT acceptance limits described b y the Approval Authority for the first two initial testing events or two out of three regular testing events administered by a vendor authoriz ed by

the Approval Authority
. The acceptance limits are laboratory mean recovery

between 2 standard deviations (SD) of the mean recovery for all approved laboratories in a given test event. Rec overies below the mean recovery minus 2 SD will fail the PT tes t event. Recoveries higher than the mean recovery plus 2 SD trigger additional evaluation, which may include one or more of the following: (1) On
- site evaluation; (2) presence of a proctor when processing PT samples during the next test event; and/or (3) s ubmission of PT microscope slides to the Approval Authority before the expiration of holding time during the next test event; Failure to submit PT slides within three weeks of PT test event when requested by the Approval Authority; Failure to m aintain records of method modifications per section 9.1.2.2 of the method; Failure to notify

the Approval Authority

of loss of key

personnel or essential equipment, change in policies or procedures that directl y affect the validity of data, or other changes affecting the capability

of the laboratory

including change in location. Laboratory Approval does not automatically

survive such changes; the Approval Authority may request an on - site or off

site evaluation and/or further proof of compliance with all applicable method requirements; Failure to submit on
- site evaluation materials and any other requested information within the time period requested by the Approval Authority ; or Failure to participate satisfactoril y in the Approval Authority Lab QA Program and demonstrate proficiency

based upon: Sample and method holding time records; anal ys t verification skills; relative quality of positive staining control and on
- going precision recovery (OPR) slides; acceptable performance of QC checks, in cluding but not limited to blind slide counts; and acceptable precision and recovery values for all method variations. Procedures for Downgrading to ``Provisionall y Approved'' Status The Approval Authority

will notify

the laboratory director or own er of its intent to downgrade after becoming aware of the situation warranting downgrading; The laboratory

director should review the problems cited, and within 30 day s of receipt of the letter, send a letter to the Approval Authority specifying imme diate corrective actions that are being taken;

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The Approval Authority

will consider the adequacy of the response and notify the laboratory

in writing of its approval status, generally within 14 day s of receipt of the laboratory's response; After the Approval Authority notifies a laboratory, the Approval Authority will post status on the Web site list of laboratories and may schedule an on
- site evaluation of the laboratory ; The laboratory

should identify

and correct its problem(s) to the Approval Authority's satisfaction within 30 days of being notified of the downgrade or have approval status suspended; A Provisionally

Approved laboratory

may continue to anal yz e samples for compliance purposes, but must identify its status as Provisionally Approved on any report; A laboratory

may request that the Approval Authority or State provide technical assistance to help identify and resolve any

problem; however, adequate performance is the laboratory's responsibility and Approval Authority

assistance should not delay the downgrading procedure. Basis for Suspending Approval Status A laboratory

may be downgraded from Approved or Provisionally Approved status to ``Not Approved'' for any of the following reasons: Repeated verification that all applicable method QC requirements have been followed, when in fact they

have not all been met; Repeated failure to document acceptable OPR values prior to anal ys is of LT2ESWTR samples; Reporting PT data from another laboratory as its own; Falsification of data or other deceptive practices including false verification that data submitted to the Data Collection and Tracking S ystem (DCTS) was generated using approved methods and met all method QA/QC criteria; Refu sal to participate in on
- site or off
- site evaluations conducted b y the Approval Authority
. Basis for Suspending Provisionally Approved Status Failure to provide a letter to the Approval Authority within 30 day s that adequately explains what immediate corrective actions were taken; Failure to identify

and correct problems in response to downgrade within 30 day s; Failure to provide accurate OPR control charts to the Approval Authority; Failure to submit valid PT results for the next two consecutive authorized PT test events within the acceptance limits specified; Continued failure to use the anal ytical methodology specified in the regulations; Failure to correct deviations identified during an on
- site evaluation within 30 day s; or Failure to provide requested demonstration, materials and documentation within 30 day s, including: acceptable matrix spike recoveries for all method variations per section 9.1.2 of the method; bench sheets, examination forms or OPR charts fo r any samples requested; remote anal ys t verification; recent positive staining control and OPR microscope slides, one of each; and blind slide counts for each anal ys t.

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Procedures for Suspension The Approval Authority

will notify

the laboratory, in writing, of its intent to suspend approval. If the laboratory wishes to request reconsideration of this decision, it should submit such a request in writing to the Approval Authority within 30 day s of receipt of the notice of intent to suspend approval. The laboratory will generall y be downgraded immediately to ``provisional approval'' in the interim while the suspension is being considered. If no request for reconsideration is filed, approval will be suspended. The request for reconsideration should be supported with an explanation of the reasons for the challenge and should be signed b y a responsible official from the laboratory such as the president/owner for a commercial laboratory , the laboratory supervisor of a municipal laboratory , or the laborato ry director for a State or Regional laboratory
. The Approval Authority

will make a decision and notify the laboratory

in writing, generall y within 30 days of receipt of the request for reconsideration. If the request is determined to be valid, the Approval Authority will take appropriate measures to reevaluate the facility and notify the laboratory , in writing, of its decision, generall y within 60 days of the reevaluation. Denial of the request will generall y result in suspension of the laborat ory's approval. Once approval is suspended, a public water s ys tem may not use the laboratory

to analyze source water samples for compliance with LT2ESWTR source water monitoring requirements. The laboratory

should notify

its clients that it is no longer approved and will not accept any more LT2ESWTR samples for anal ys is. Upgrading or Reinstatement of Approval Subject to the availability of resources, the Approval Authority will consider written requests from the laboratory to seek upgrading or reinst atement of approval. Requests should state the reasons why the laboratory should regain its approval status. The laboratory should demonstrate that all deficiencies have been corrected and successfull y complete two consecutive authorized PT test events within acceptance limits for Provisionally Approved laboratories or three consecutive authorized PT test events within acceptance limits for suspended laboratories. The authorized PT test events being described here are those submitted to all laboratories in the Lab QA Program, not special issue blind samples purchased independentl y from the vendor. The laboratory

should provide evidence why the reasons for downgrading or suspension are no longer applicable and explain its technical competence. Acceptable demonstration of technical competence may

include an on
- site evaluation and/or any other measure the Approval Authority deems appropriate. The Approval Authority will consider compliance history, corrective actions implemented by

the laboratory , effectiveness of corrective actions, and professional judgment of the Approval Authority. Grievances Laboratories with grievances during the authorized PT events or regarding participation in the L ab QA Program should immediately contact the Program Manager at th e Approving Authority and try to remedy

the problem. When the laboratory feels they

have not gotten immediate or satisfactory results, they should contact the supervisor at the Approving Authority
. The management at the Approving Authority will work with the Program Manager to quickl y

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address grievances. A final decision for all grievances will be made generally within 30 day s of contacting the Approving Authority
. Request for Comment The EPA is soliciting comments on this notice to: 1. Evaluate whether the proposed collection of information is necessary for the proper performance of the functions of the Agency , including whether the information will have practical utility ; 2. Evaluate the accuracy

of the Agency's estimate of the burden of the proposed collection of information, including the validity of the methodology

and assumptions used; 3. Enhance the quality , utility, and clarity of the information to be collected; 4. Minimize the burden of the collection of information on tho se who are to respond, including through use of appropriate automated electronic, mechanical, or other technological collection techniques or other forms of information technology, e.g., permitting electronic submission of responses; and 5. Consider any

necessary

changes to the Lab QA Program. As an example, EPA is particularl y interested in comments from States regarding the potential for their laboratory programs to assume any/ all responsibility for the approval and oversight of LT2ESWTR laboratories , including comments on the appropriate timeframes for such. The Agency also welcomes comments regarding the appropriateness of turning to commercial PT providers as the source of PT samples for laboratories, in lieu of the PT program currentl y administere d by the Agency
. Burden Statement: The burden estimate for the Lab QA Program information collection includes all the burden hours and costs required for gathering information, and developing and maintaining records associated with the Lab QA Program. An estimated 65respondents will participate in an average of 4.4 responses per ye ar to include: anal ys is and reporting of PT samples three times per year, application for initial or re
- audit once every three years, off

- site re
evaluation activities once every three years, and on
- site evaluation once every three years. A small subset of laboratories will perform follow
- up activities based on inadequate QA/QC, failed OPRs, incomplete records, delayed communication t o EPA or poor PT results. A few laboratorie s perform more than one method version and will anal yz e an additional set of PT s amples three times per ye ar. The total annual public reporting and recordkeeping burden for this collection of information is estimated to be 4843 hours at a cost of $269,800. 40. The average hours and cost per response for the average of 4.4 responses per year are 16.9 hours and $943.36, respectivel y. These estimates assume that laboratories participating in the Lab QA Program have the necessary

equipment needed to conduct the analy ses. Therefore, there are no start
- up costs. The estimated total annual capital cost is $0.00. The total estimated Operation and Maintenance (O&M) costs is $141,929.00. The I CR provides a detailed explanation of the Agency's estimate, which is on ly briefly summarized here: Estimated Total Number of Potential Respondents: 65. Frequency

of Response: Annual. Estimated Total Average Number of Responses for Each Respondent: 4.4. Estimated Total Annual Burden Hours: 4843 hours.

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Estimated Total Annual Costs: $411,729.40. This includes an estimated burden cost of $269,800.40 and an estimated cost of $141,929.00 for capital investment or maintenance and operational costs. Are There Changes in the Estimates From the Last Approval? Changes in burden have occurred due to inflation, re
- evaluation of hours for tasks, and improved demonstration of capability. Inflation has increased all operation and maintenance and labor costs accordingl y. The increase in the respondent universe ha s increased the overall burden costs for the respondents. EPA's original estimates for hours to participate and maintain the Lab QA Program were made before the program began. Because the program has matured and several ye ars of QC data have been collected , the burden has changed for performing improved and refined procedures. The burden for some tasks has been estimated and will be re
- evaluated as the program progresses. EPA has added the preceding section entitled ``Clarification of Basis and Procedures for Downgrading/Suspending Approval for Laboratories for the Anal ys is of Cryptosporidium in Water Under the Long Term 2 Enhanced Surface Water Treatment Rule.'' Some approved laboratories may have to undertake additional activities to demonstrate continued acceptable performance to EPA, which may increase the burden of participation in the Lab QA Program for those laboratories. EPA estimates that nine laboratories per ye ar may have to undertake additional activities to demonstrate acceptable performance to EPA. These estimates will be corrected as the program continues. What is the Next Step in the Process for This ICR? EPA will consider the comments received and amend the ICR as appropriate. The final ICR package will then be submitted to OMB for rev iew and approval pursuant to 5 CFR 1320.12. At that time, EPA will issue another Federal Register notice pursuant to 5 CFR 1320.5(a)(1)(iv) to announce the submission of the I CR to OMB and the o pportunity to submit additional comments to OMB. If you have any questions about this I CR or the approval process, please contact the technical person listed under FOR FURTHER INFORMATION CONTACT. Dated: February

19, 2009. Cy nthia C. Dougherty, Director, Office of Ground Water and Drinking Water. [FR Doc. E9
- 4009 Filed 2
- 24
- 09; 8:45 am] BILLING CODE 6560
- 50
- P

### E
- 1 

# APPENDIX 

# E Federal Register Notice : Laboratory

Quality Assurance Evaluation Program/ In formation Collection Request [Published XX XX , 20 13]

### E
- 2

### F
- 1 

# APPENDIX 

# F Comments on XX XX, 2013 Federal Register Notice : Laboratory

Quality Assurance Evaluation Program/ In formation Collection Request

### F
- 2

### G
- 1 

# APPENDIX 

# G Application for Assessment of Cryptosporidium Laboratory QA

### G
- 2 Office of Ground Water and Drinking Water Standards and Risk Management Division

[Date]
Dear : Your laboratory is due to be evaluated under [State] Program for Analysis of Cryptosporidi u m . The evaluation will include personnel qualifications, quality control, equipment maintenance, record keeping, sample processing and analysis procedures based on the January 2012 version of Method 1623.1 or the December 2005 version of Method 1623. Here is the process: **Step 1 )**  **Submit the attached application form and package** within 30 days of receipt of this letter. Electronic copies are appreciated. **Step 2 )**  **Coordinate shipment of an OPR slide and the associated positive control slide**  with [Cert Officer name and email or the contractor operating on behalf of EPA, Dr. Leah Villegas, CB&I, villegas.leah@epa.gov or 513
- 569
- 7886 ]
. **Step 3 )**  **Order on**  **- site audit package including blind samples and analyst v**  **erification slides**  spiked with Cryptosporidium and Giardia from Wisconsin State Laboratory of Hygiene, 608
- 224
- 6260. These will be used in presence of an auditor; therefore, on
- site evaluation date should be within product expiration. Submit the bench sheets and examination forms for the blind samples and associated method blank and OPR samples, and analyst verification slides with slide examination forms to [ Cert Officer name and email ]
. **Step 4 )**  **Schedule an online**  **analyst skills evaluation**  for all analysts with [ Cert Officer name and email or the contractor operating on behalf of EPA, Dr. Leah Villegas, CB&I, villegas.leah@epa.gov or 513
- 569
- 7886 ]
. **Step 5 )**  **Schedule an on -**  **site visit Step 6** **) Provide**  **written responses** to any deficiencies found, or recommendations made, by the certification officer
. We look forward to working with you. If you have any questions, please contact me at 

### [certification officer]

. Sincerely,

Certification Officer and affiliation

cc: Enclosures: Application Package and Form Checklists in Supplement 2 (http://water.epa.gov/scitech/drinkingwater/labcert/index.cfm)

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- 3 

## Ap **plication Package for As**  **sessment of**  **Cryptosporidium Laboratory**  **Quality A** **ssurance Submit electronic package to:**  [Cert Officer email] **Submit any**

**necessary**

**hard copies to:** [Cert Officer Name]

[Address] [Phone] 

### Step 1: **Submit all requested information** Please submit all requested elements in one package organized as follows. Your application will be evaluated for completeness
. **1.** Completed Audit Application Form **2.** Up to date Standard Operating Procedures (SOPs) for the following: **a.** Performance of each Method step including: sample spiking, filtration, elution, concentration, purification, slide preparation, sample staining and examination (for each method version, where applicable) **b.** Reagent preparation **c.** Cleaning practices **d.** Corrective action procedures for failing to meet OPR, method blank, staining controls, sample acceptance and analyst verification criteria **e.** Sampling procedures to be followed by field or utility personnel **f.** Procedures for data recording, checking manual calcu lations, and checking accuracy of all data transcriptions **g.** Procedures for data recording and electronic storage of data, including checking for accuracy of data entry and backup of stored data **3.** Training records for all analysts/technicians **4.** OPR control chart including at a minimum the last 20 OPR samples processed **5.** MS control chart including at a minimum the last 20 MS samples processed **6.** Submit two data packages from the last 12 months , include a positive result if avail able . Include all supporting documentation from the field sample, matrix spike, method blank and positive control slide. **7.** NELAP certificate (as applicable)
**Step 2**  **: Submit slides for review**  An off
- site technical auditor will review one recent OPR and associated positive staining cont rol slide. Contact [Cert Officer Name and email or the contractor operating on behalf of EPA, Dr. Leah Villegas, CB& I, villegas.leah@epa.gov or 513
- 569
- 7886 ] to schedule evaluation of slides. This review may occur prior to or following the on
- site audit . The slides prepared for the review during the audit in Step 5 may be requested to fulfill this submission. **Step 3: Order**  **on -**  **site audit package** Order an on
- site audit package from Wisconsin State Laboratory of Hygiene (608
- 224
- 6260), or equivalent vendor. Each package consists of 2 50 mL blind samples, 2 vials of artificial matrix, 2 IMS control samples, and 2 analyst verification slides. One sl ide is required per analyst and a back up slide. Additional slides may be added for a fee. These will be used in presence of an auditor; therefore, evaluation date should be within product expiration. Bench sheets and examination forms for the blind samp les, and associated method blank and OPR samples should be submitted to [Cert Officer Name and email]
. Submit the analyst verification slides and associated examination forms , if requested. **Step 4: Schedule internet analyst evaluation** 

### G
- 4

Contact [ Cert Officer Name and email the contractor operating on behalf of EPA, Dr. Leah Villegas, CB& I, villegas.leah@epa.gov or 513
- 569
- 7886 ] to schedule internet analyst evaluation for each analyst. A computer with intern et connection is needed to complete the session. **Step 5**  **: Prepare fresh OPR and positive staining control for review during the audit**  **Step 6: Evaluation**  The laboratory will receive a report detailing all audit findings. The laboratory should provide complete written responses to any deficiencies or recommendations identified in the report within 60 days. Laboratory status for continued approval will be based on submission of acceptable respo nses, proficiency test results, the quality of the positive control and OPR slide, slide counts, on
- site evaluation, and recovery values for blind samples initiated during audit

### G
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## Cryptosporidium **Laboratory A**  **udit Application Form Part 1. Laboratory**  **Information**

**Laboratory**

**Name: Ad**  **dress: City**  **: State:**  **Zip: Contact Person:**  **Title: Telephone:**  **Fax: Email address:**  **Ty pe of laboratory** 

**(check one): Commercial**  **Utility State**  **Ac ademic**  **Other Method used in the Laboratory**  **: December 2005 Method 1623**  **January 2012 Method 1623.1**  **Number of field sample**  **s y our laboratory**  **is analy zing**  **per month using Method 1623 and/or 1623.1**  **: Date of Previous Audit:**  **Date of Initial Approval:** 

### Part 2 **. Personnel List**

**Name of Current Anal**  **ys t and**  **Technicians Current position**  **(Principal Analy st, Anal**  **ys t, or Technician)**  **Evaluated during**  **Previous Audit**  **or Documentation Submitted to EPA** 

**(Yes/No)**

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- 6

**Part 3 . Method**  **and Equipment Information:**  **Versions of Method for which the lab is s**  **eeking e**  **valuation Method**  **Procedure Ke**  **y Equipment**  **Manufacturer/Model C**  **heck all that apply**  **Provide manufacturer and model for relevant pieces of equipment Filtration**  **I ndicate the volume filtered for each**  Cubitainer Pall Envirochek® HV Pump I DEXX Filta - Ma x ® Flow control valve Other (describe) F low meter or graduated container **Elution** Wr ist action shaker Laboratory shaker and side arms Filta - Max

® wash station Filta - Max ® Manual station Other (describe) Filta - Max ® Automatic station **Concentration** Centrifugation Centrifuge - 1500 X G, swinging - bucket centrifuge for 15 mL - 250 - mL tubes Filtration through membrane Other (describe) Concentrator apparatus (Filta - Ma x only)

**Purification** Dynabeads ® Crypto Flat - sided sample tubes Dynabeads ® C G - combo Sample mixer/rotator for 10 - mL tubes Other (describe) Magnetic particle concentrator for 10 - mL tubes Magnetic particle concentrator for 1.5 - mL tubes **Staining and Examination** Wa terborne AquaGlo TM Microscope - Epifluorescence/ differential interference contrast (HMO or DIC) microscope with stage and ocular micrometers Wa terborne Crypt - a - Glo TM Wa terborne Giardi - a - Glo TM 20X to 100X objectives Meridian Merifluor ® Excitation/band pass microscope filters for fluorescein isothiocyanate (FITC) assay (provide specifications) BTF EasyStain TM

### G
- 7 **Method Procedure**  **Ke y**  **Equipment Manufacturer/Model**  Other (describe) Excitation/band - pass filters for 4',6 - diamidino - 2 - phenylindole (DAPI) assay (provide specifications) **Other** Descriptions of “other” method steps and other comments: Refrigerator for sample storage Refrigerator for reagent storage **The above information is complete and accurate to the best of my knowledge.** 
**Name and Signature of**  **Laboratory Director or Designee**  **Date**

### G
- 8 

## Checklist A **– Method 1623/1623.1**  **Audit Package and Data Review Laboratory** 

**Name Name and A**  **ffiliation of Evaluator Date of Evaluation** 
Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the con sistency and reliability of results. Adapted from other federal programs for the purposes of the Cryptosporidium Laboratory QA Eva luation Program , GLP includes personnel, equipment, and standard operating procedures appropriate for the program

. 

### Item to be Evaluated **Reference*** **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK 1**  **Quality Assurance**  1.1 Is documentation (e.g., resume, sample list) available for all Method 1623/ 1623.1 staff? 9.1 9.1 1. Requirement GLP 1.1.1 Have technicians/analysts analyzed the

required number of samples using Method 1623 or 1623.1? 22.2 22.2 1.1 - 1.3 Requirement GLP 1.2 Are employee training records available and up to date? 9.1 9.1 1.7 Critical GLP 1.2.1 Have all analysts documented that they have read and understood the QA Plan and SOPs? - - 1.7 Critical GLP 1.3 Is the laboratory performing analyst verification monthly and does the lab have corrective action procedures in place if criteria are not met? 10.6 9.10 7.1.9 Requirement 1.3.1 If the laboratory has only one analyst, is the analyst demonstrating analyst verification through comparison with photo libraries or repetitive counts ? 10.6.4 9.10.4 7.1.9.5 Recommendation
1.4 Does the quality assurance plan address requirements for Cryptosp oridium analysis under LT2ESW TR ? Critical 1.5 Have acceptable initial precision and recovery analyses been performed for each version of the me thod the laboratory is using? 9.1.2.1.1 9.2 7.1.4 Requirement

### G
- 9

**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 1.6 Of the field/PT samples reviewed, is each field/PT sample associated with an acceptable method blank? 9.6.1 9.7 7.1.2 7.1.5.1 Requirement 1.6.1 We re all method blanks (MB) evaluated without contamination? 9.6.2.1 9.7.2 7.1.5 Requirement # MB reviewed : 1.6.2 We re the same lots of reagents (elution, IMS, and staining) used for the method blank and the associated field/PT samples? - - 7.1.5.3 Critical
1.6.3 Is method blank analyzed prior to the analysis of field/PT samples? 9.6 9.7 7.1.5.2 Requirement 1.7 Is each field/PT sample associated with an acceptable ongoing precision and recovery (OPR) sample? 9.7 9.8 7.1.2 7.1.6.1 Requirement
1.7.1 Wh at percentage of OPR samples evaluated met the recovery criteria? 9.7.3 Table 3 Table 4 9.8.3

Table 3 Table 4 7.1.6.2 # OPR reviewed:
1.7.2 We re the same lots of reagents (elution, IMS, and staining) used for the OPR and the associated field/PT samples ? - - 7.1.6.1 Critical 1.7.3 Is OPR analyzed prior to the analysis of field/PT samples? 9.7.1 9.8.1 7.1.6.2 Requirement
1.7.4 Does the laboratory maintain control charts of OPR results? 9.7.6 9.1 9.8.3

9.12.1 Table 2 7.1.7 1623 Recommendation 1623.1 Requirement 1.7. 5 Wh at is the mean and relative standard deviation (RSD), or standard deviation, of the recoveries of the OPR samples included in the control chart? 9.4.3 Table 3 Table 4 9.5.3

Table 3 Table 4 7.1.6.2 QC Criteria Mean: RSD: 1.8 We re matrix spike (MS) samples analyzed at the minimum frequency of 1 MS per 20 (up to and including) field samples from each source? 9.1.8 9.6.1 7.1.2 7.1.10.2 Requirement # MS reviewed: 1.8. 1 We re MS sample volumes within 10% of their associated field samples’ volumes ? 9.5.1 9.6.2 7.1.10.3 Requirement 1.8. 2 We re MS samples analyzed at the same time and using the same method variation as their associated field samples? Table 2 Table 2 7.1.10.1 Requirement

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**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 1.8. 3 Wh at is the mean and relative standard deviation of the MS samples reviewed? Tab le 3 Table 4 Table 5 Table 3

Table 4 Table 6 - QC Criteria Mean: RSD: 1.8. 4 Does the laboratory maintain control charts of MS results? 9.5.1.4 9.1 9.6.2.3

9.12.2 Table 2 7.1.10.4 1623 Recommendation 1623.1 Requirement 1.9 We re OPR samples spiked with 100 - 500 organisms? 9.7 9.8 7.1.6.1 Requirement 1.10 Does the laboratory perform IMS controls and ma intain IMS control charts? If not, how do they troubleshoot low recoveries? 9.7.5.3 9.8.7.3 9.13 - Recommendation 1.1 1 Does the laboratory have an adequate record system for tracking samples, including unique ID, from coll ection through log - in, analysis, and data reporting? - - 8.0 Critical GLP 1.1 2 Is the laboratory using the Method December 2005 version of Method 1623 or Method 1623.1 for LT2 samples? Requirement **2 Data Recording Procedures**  2.1 Is shipping information complete, i.e., time/date of sample collection, sampler's name, time/date of sample receipt, receiver's initials, sample condition? 8.1.3 8.1.3 8.5 Requirement
2.1.1 We re all samples evaluated received at ≤20 **°** C and not frozen? 8.1.3 8.1.3 6.3.3 Requirement 2 .2 Do sample numbers on the chain of custody ma tch the sample numbers on the report forms? - - - Requirement 2 .3 Are current Method 1623/ 1623.1 bench sheets used to record sample processing data? - - 8.2 Recommendation 2.4 Are all primary measurements during each step recorded, including all raw data used in calculations? 9.1.2.2.5 9.3.5 8.0 Requirement 2. 5 Technician/analyst, d ate , and time of elution is recorded? 12.2.6.2.1 12.2.7.1 12.3.2.1 8.7 Requirement 2. 6 Technician/analyst, d ate , and time of slide preparation is recorded? 13.3.3.11 13.3.3.11 8.7 Requirement

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**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 2. 7 Technician/analyst, date, and time of staining is recorded? 14.10 14.10 8.7 Requirement 2. 8 Are batch and lot numbers of reagents used in the analysis of the sample recorded? - - 8.7 Critical 2. 8.1 Lot number for the IMS kit is recorded? - - 8.7 Critical 2. 8.2 Lot number of the staining kit is recorded? - - 8.7 Critical 2.8.3 Lot number of the spiking suspensions is recorded? - - 3.21.4 8.7 Critical 2

. 9 Spike value recorded for all spiked samples? - - 3.21.4 8.6 Requirement 2.1 0 Are Method 1623/ 1623.1 Cryptosporidium Slide Examination forms used to record sample examination results? 15.2 15.2 8.2 Requirement 2.1 1 Name of examining analyst is recorded? 15.2.6 15.2.6 8.7 Requirement 2.1 2 Date and time of sample examination is recorded? 15.2.4 15.2.4 8.7 Requirement 2.1 3 Are calculations of final concentrations and recoveries complete and correct? - - - Requirement 2.14 Is the size of the cysts and oocysts reported to the nearest 0.5 **μ** m? 15.2.2.3 15.2.3.3 15.2.2.4

15.2.3.4 5.4.9.4

5.4.10.4 Requirement 2. 15 Is each reported positive organism detected in a field sample characterized and recorded? 15.2 15.2.2.1 15.2.3.1 5.4.9.1

5.4.10.1 Requirement 2.16 Do values recorded on the data sheets match the values reported to the client? - - 8.1 Requirement 2.17 Are mistakes on all forms crossed out with a single line, initialed, and dated? - - 8.2 Critical 2.18 Are data always legible and recorded in pen? - - 8.2 Critical 2.19 Wa s the final report reviewed by QA manager, lab director or an individual other than the analyst? - - 8.1 Critical 2.2 0 Do records demonstrate each analyst's characterization of 3 oocysts and 3 cysts from positive control for each microscopy session? 15.2.1.1 15.2.1.1 5.4.6 Requirement 2.2 1 Data shows that no more than 0.5 mL of pellet was used per IMS? 13.2.4 13.2.3 5.2.3 8.6 Requirement

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**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK 3**  **Holding Times –**  **Method 1623.1**  3.1 Is sample elution initiated within 96 hours of sample collection or field filtration? 8.2.1 Table 1 8.2.1

Table 5 6.4

8.7 Requirement 3.2 Are sample elution, concentration, and purification steps completed in one work day? 8.2.2 Table 1 8.2.2

Table 5 6.4

8.7 Requirement 3.3 Are slides stained within 72 hours of application of the purified sample to the slide? 8.2.3 Table 1 8.2.3

Table 5 6.4

8.7 Requirement 3.4 Are stained slides read and confirmed within 7 days of staining? [Section 8.2.4 and Table 5 ] 8.2.4 Table 1 8.2.4

Table 5 6.4

8.7 Requirement **4 Spike enumeration procedures**  4.1 Source of flow cytometry - enumerated spiking suspensions. - 11.2
- 4.2 If 50 - L samples are analyzed, what positive control procedure does the laboratory follow for OPR and MS samples: (A) spike entire 50 L, (B) spike and filter 10 L before filtering 40 L, or (C) filter 40 L before spiking and filtering 10 L. - - 7.1.10.3 The following items below are optional if the laboratory is NELAC certified. If the laboratory opts to provide NELAC certific ation, complete the box below by entering the NELAC certification number and date. Provide copy of certification. **NELA C Certification Number:**  **Certification Date :**  **5 Laboratory** 

**Equipment and Supplies 5.1**  **Reagent -**  **grade water testing** 5.1.1 Is reagent water tested monthly for conductivity and total chlorine residual? - - 4.3.1 Critical GLP 5.1.1.1 We re the results for the above

parameters acceptable? Total chlorine residual not greater than 0.1 mg/L, conductivity not greater than 2 μmhos/cm? - - 4.3.1 Critical GLP
5.1.2 Has the reagent water been tested annually for metals

– Pb, Cd, Cr, Cu, Ni, Zn? - - 4.3.1 Critical GLP

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**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 5.1. 2.1 We re the results for the metals

testing acceptable; each metal not greater than 0.05 mg/L and collectively not greater than 0.1 mg /L? - - 4.3.1 Critical GLP
5.1.3 Is reagent water tested monthly for heterotrophic plate count? - - 4.3.1 Critical GLP 5.1.3.1 Are the results for the

heterotrophic plate count acceptable, < 500 CFU/mL? - - 4.3.1 Critical GLP 5.1.4 Is still or DI unit maintained according to

manufacturer's instructions? - - 4.3.3 Critical GLP **5.2 pH meter**  5.2.1 Accuracy ± 0.1 units, scale graduations,

0.1 units? - - 3.1.1 Critical GLP 5.2.2 Is a record maintained for pH

m easurements and calibrations? - - 3.1.4 Critical GLP 5.2.3 Is pH meter standardize d each use

period with pH 7, 4 or 10 standard buffe rs (selection dependent upon desired pH)? - - 3.1.4 Critical GLP 5.2.4 Are all pH buffers dat ed when received

and opened, and discarded before expiration date? - - 3.1.5 Critical GLP **5.3 Balances (top loader or pan balance)**  5.3.1 Are balance calibrations verified monthly using ASTM Class 1, Class 2 or Class 3 weights or weights traceable to Class 1, Class 2, or Class 3 weights, or equivalent? Non - reference weights should be calibrated every six months with reference weights. - - 3.2.2 Critical GLP

5.3.2 Is correction data and Certificate of Traceability available for weights? - - 3.2.3 Critical GLP 5.3.3 Is preventative maintenance conducted yearly at a minimum? - - 3.2.4 Recommendation GLP **5.4 Temperature recording**  **device**

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**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 5.4.1 Are calibration of thermometers checked annually (dial thermometers quarterly) at the temperature used against a reference NIST thermometer or equivalent? 8.1.4 8.1.4 3.3.2 Requirement GLP 5.4.2 Is the sample storage refrigerator able to ma intain temperature of 1 to 10°C? - - 3.7.1 Critical GLP **5.5 Micropipetters**  5.5.1 Have micropipetters been calibrated within the past year? 9.2.1 Appendix A 3.8.2 Requirement GLP **5.6 Centrifuge**  5.6.1 Is a maintenance contract in place or internal maintenance protocol available ? 9.1 9.1 3.15.5 Critical GLP 5.6.2 Is the centrifuge calibrated yearly? - - 3.15.5 Critical GLP **5.7 Au**  **toclave** 5.7.1 Are date, contents, sterilization time and temperature, and technician initials recorded for each cycle? - - 3.5.3 Critical GLP 5.7.2 Is a maximum registeri ng thermometer or continuous monitoring device used during each autoclave cycle? - - 3.5.5 Critical GLP 5.7.3 Is automatic timing mechanism checked with stopwatch quarterly? - - 3.5.6 Critical GLP 5.7.4 Are spore strips or am pules used monthly to confirm sterilization? - - 3.5.5 Critical GLP **6 Quality** 

**A ssurance**  **Manual** 6.1 Does the laboratory have a formal QA laboratory plan prepared and ready for examination? 9.1 9.1 4.5.1 Requirement 6

. 2 Is a laboratory organization chart or other information available listing staff organization and responsibilities? Does it identify the QA manager and lab director? 4.5.2 Recommendation
6.2.1 Is the QA manager separate from the lab director? - - 4.5.2 Recommendation GLP

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**Item to be Evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 6.3 Does the laboratory have a schedule and/or procedure for all preventative maintenance of equipment ? - - 4.5.3 GLP 

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## Checklist B **- Laboratory**  **SOP Review Laboratory** 

**Name Name and A**  **ffiliation of Evaluator Date of Evaluation** 

Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the con sistency and reliability of results. Adapted from other federal programs for the purposes of the Cryptosporidium Laboratory QA Evaluation Program , GLP includes personnel, equipment, and standard operating procedures appropriate for the program

. 

### Item to be Evaluated **For each item, does the SOP specify:** 

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  **1 Sample Spiking**  1.1 The suspension vial is vortexed for 30 seconds or per manufacturer’s instructions ? 11.4.3.1.2 11.2.3.2 - Method Procedure 1.2 The carboy used for the method blank is randomly selected from carboy stock to check efficacy of cleaning system or disposable carboys are used for all samples? - - 7.1.5.3 Critical
1.3 The details of the suspension vial rinse, including volumes ? 11.4.3.1 11.2.3 - Method Procedure 1.4 Acceptable sample spiking procedures, including issues not noted in items 1.1 through 1.3? Critical GLP **2 Filtration/Elution 2.1**  **Envirochek**® **HV filtration** 2.1.1 The flow rate is maintained at approximately 2 L/ min? 12.2.1.2 12.2.1.2 - Method Procedure 2.1.2 The volume filtered is measured using a flow totalizer or calibrated carboy? 12.2.4.2 12.2.4.2 - Requirement 2.1.3 The sample is stirred during filtration? 12.2.4.1 12.2.4.1 - Method Procedure 2.1.4 The details of the carboy rinse after filtration including volume? 12.2.4.5 12.2.4.6 - Method Procedure

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  2.1.5 Appropriate maintenance and cleaning procedures? - - - Critical 2.1.6 Acceptable Envirochek® filtration procedures, including issues not noted in items 2.1.1 through 2.1.5? Critical GLP **2.2 Envirochek ®**  **HV capsule filter elution**  2.2.1 M easurement of the volume of the elution buffer used or that the volume covers the me mbrane? 12.2.6.2.2 12.2.8.2 - Method Procedure 2.2.2 The speed that samples are shaken? 12.2.6.2.3 12.2.8.3 - Method Procedure 2.2.3 The dispersant is added to the sample as per Method 1623.1? 12.2.7 - 1623 Recommendation 1623.1 Requirement 2

. 2.4 The samples are shaken three times for 5 minutes each time, and each in a different orientation? 12.2.6.2 12.2.8 - Method Procedure 2.2.5 Procedures for filter capsule rinse and addition of rinsate to the centrifuge bottle? 12.2.6.2.8 12.2.8.8 - Method Procedure 2.2. 6 Acceptable Envirochek® capsule filter elution procedures, including issues not noted in items 2.2.1 through 2.2. 5 ? Critical GLP **2.3 Filta**  **- Max** **® filtration**  2.3.1 The flow rate is maintained at ≤ 4 L per minute for Filta - Max ® ? 12.3.1.1.3 12.3.1.1.3 - Method Procedure 2.3.2 The volume filtered is measured using a flow totalizer or calibrated carboy? 12.3.1.5.2 12.3.1.5.2 - Requirement 2.3.3 Appropriate maintenance and cleaning procedures? [Section 12.3.4 ] 12.3.4 12.3.4 - Requirement 2.3.4 Acceptable Filta - Max ® filtration procedures, including issues not noted in items 2.3.1 through 2.3.3? Critical GLP **2.4 Filta -**  **Max ®**  **filter wash station elution** 2.4.1 The use of PBST to elute the filter? 7.4.2.4 7.6.2.4 - Method Procedure

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  2.4.2 The am ount of PBST used for each wash? (approx. 600 mL ) 12.3.2.2 12.3.2.2 - Method Procedure 2.4.3 T he plunger is moved up and down 20 times during the first wash? 12.3.2.2.1 h 12.3.2.2.1

h - Method Procedure 2.4.4 T he plunger is moved up and down gently to avoid generating excess foam? 12.3.2.2.1 h 12.3.2.2.1

h - Method Procedure 2.4.5 That d uring the second wash the plunger is moved up and down 10 times? 12.3.2.2.2 b 12.3.2.2.2

b - Method Procedure 2.4.6 The instructions for cleaning the wash station between samples? 12.3.4.2 12.3.4.2 - Requirement 2.4.7 The housing is rinsed after filter is removed and the rinse is included in the sample volume? 12.3.2.2.1 d 12.3.2.2.1

d - Method Procedure 2.4.8 Acceptable Filta - Max ® filter wash station elution procedures, including issues not noted in items 2.4.1 through 2.4.7? Critical GLP **3 Concentration 3.1**  **Filta -**  **Max ®**  **filter sample concentration (as an alternative or in addition to Section 3.2)** 3.1.1 T he force of the vacuum is maintained below 30 cm Hg? NOTE pg 43 NOTE

pg 34 - Method Procedure 3.1.2 That concentration is performed after each of the washes? 12.3.2.2.1 j 12.3.2.2.1

j - Method Procedure 3.1.3 T he sample is concentrated so that some liquid remains above the filter ( enough to cover the stir bar about half -way)? 12.3.3.2.1

c 12.3.3.2.1

b - Method Procedure 3.1.4 T he stir bar and concentration tube are rinsed after each concentration and the liquid added to the concentrate? 12.3.3.2 12.3.3.2 - Requirement 3.1.5 T he filter membrane is washed twice with 5 mL of PBST each time ? 12.3.3.2.3 12.3.3.2.3 - Method Procedure 3.1.6 Acceptable Filta - Max ® filter sample concentration procedures, including issues not noted in items 3.1.1 through 3.1. 5 ? Critical GLP **3.2 Envirochek** ® **HV and Filta**  **- Max** **® filter sample centrifugation** 

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  3.2.1 The sample is centrifuged at 1500 x G (maximum 2000 x G) using a swinging bucket rotor? 13.2.1 including NOTE 13.2.1

including NOTE - Method Procedure 3.2.2 Instructions to ensure the centrifuge tubes are properly balanced prior to centrifugation? - - 3.15.4 Critical 3.2.3 The sample is centrifuged for 15 minutes with start time beginning when centrifuge reaches the required speed ? 13.2.1 13.2.1 - Method Procedure 3.2. 4 The centrifuge is slowly decelerated at the end without using the brake ? 13.2.1 13.2.1 - Method Procedure 3.2. 5 Acceptable Envirochek® HV and Filta Max ® filter sample centrifugation procedures, including issues not noted in items 3.2.1 through 3.2. 4 ? Critical GLP **4 Purification and Slide Preparation**  4.1 The centrifuged sample supernatant is aspirated no lower than 5 mL of supernatant above every 0.5 mL of the pellet or portion of 0.5 mL pellet? 13.2.2 13.2.2 13.2.3 5.2.2

5.2.3 Requirement 4.1.1 The type and internal diameter of pipette used for aspiration of supernatant? - NOTE pg 37 - Recommendation 4.1.2 The rate of aspiration (i.e., mL/ min or pressure of the vacuum)? - 13.2.2 - Recommendation 4.2 The tube is vortexed vigorously until pellet is completely resuspended ? 13.2.3 13.2.2.1 - Method Procedure 4.3 Appropriate procedures for dividing pellets greater than 0.5 mL into subsamples and the analysis of the subsamples? 13.2.4 13.2.3 - Critical 4.4 No more than 0.5 mL of pellet is used per IMS? 13.2.4 13.2.3 5.2.3 Method Procedure 4.5 The resuspended pellet volume is quantitatively transferred to the flat - sided tube (2 rinses) including the determination of the rinse volumes? 13.3.2.1 13.3.2.1 - Method Procedure 4.6 SL - Buffer A is used at room temperature or that it is checked for precipitate before use? NOTE pg 47 NOTE

pg 39 3.17.2 Method Procedure 4.7 The volume of 10x SL - Buffer A is 1 mL ? 13.3.1.2 13.3.1.2 5.2.5 Method Procedure

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  4.8 The volume of 10x SL - Buffer B is 1 mL ? 13.3.1.3 13.3.1.3 5.2.5 Method Procedure 4.9 Instructions for thorough resuspension of IMS beads prior to addition to the flat - sided tube ? 13.3.2.2 13.3.2.4 13.3.2.2

13.3.2.4 - Method Procedure 4.10 100 μL of Cryptosporidium and Giardia beads are used? 13.3.2.3 13.3.2.5 13.3.2.3

13.3.2.5 5.2.5 Method Procedure 4.11 The flat - sided tube is rotated at 18 rpm for 1 hour at room temperature? 13.3.2.6 13.3.2.6 - Method Procedure 4.12 Wh ich magnetic concentrators, MPC ®

- 1 or MPC ®

- 6, are used? Method Procedure 4.13 The placement of the flat - sided tube in the magnet and the rock technique and time ? 13.3.2.9 13.3.2.8 13.3.2.9 - Method Procedure 4.14 The sample is quantitatively transferred from the flat - sided tube to the microcentrifuge tube (2 rinses) including rinse volumes? 13.3.2.13 13.3.2.14 - Method Procedure 4.15 The flat - sided tube is allowed to sit one minute after each transfer to accumulate residual sample, then the residual is transferred to microcentrifuge tube? 13.3.2.13 13.3.2.14 - Method Procedure 4.16 The magnet is in the vertical position in the MPC ®

- S? - 13.3.2.13 - Method Procedure 4.17 The beads are rinsed with PBS while inside the microcentrifuge tube? 13.3.4 13.3.2.17 - 1623 Recommendation 1623.1 Requirement 4.18 Standard NaOH (5 μ L, 1N) and standard HCl (50 μ L, 0.1N) are used? NOTES pg 49 - 50 NOTES

pg 42 3.17.5 Requirement 4.19 The sample is vortexed vigorously for 50 seconds immediately after the addition of acid and 30 seconds after the sample has set for 10 minutes at room temperature? 13.3.3 13.3.3 - Method Procedure 4.20 The magnet is in the slanted position in the MPC ®

- S for dissociation steps ? - 13.3.3.6 - Method Procedure
4.21 A second dissociation is performed ? 13.3.3.10 13.3.3.10 5.2.4 Requirement

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  4.22 Wh en the second dissociation is performed, the laboratory: A) uses a second slide, or B) adds the additional volume to the original slide? 13.3.3.10 13.3.3.10 13.4.5 - Circle one: A B 4.23 The volume and the timing of the NaOH addition to the wells ? 13.3.3.8 13.3.3.8 - Method Procedure 4 .2 4 Wh en the slides are dried (e.g., room temperature or slide warmer), the laboratory: A) uses room temperature , or B) uses 35 o to 42 o C , or C) follows manufacturer’s instructions? 13.3.3.12 13.3.3.12 - Circle one: A B C
4.25 If the laboratory has more than one option specified for slide drying, are criteria included for when each option will be used? - - 5.3.1 Recommendation 4.26 That positive and negative staining controls are prepared at the same time the slides are prepared ? 14.1 14.1.3 - Requirement 4.2 7 Acceptable sample purification and slide preparation procedures, including issues not noted in items 4.1 through 4.2 6 ? Critical GLP **5 Sample S taining**  5.1 Wh ich stain to use and to follow manufacturer’s instructions for FITC stain application? 14.2 14.2 5.3.2 Method Procedure 5.2 The slides are incubated in a humid chamber in the dark at room temperature for approximately 30 minutes or per manufacturer’s directions? 14.3 14.3 5.3.3 Method Procedure 5.3 The working DAPI stain is prepared the day it is used? 7.7.2 7.9.2 3.19.2 Method Procedure 5.4 The stock DAPI is stored at 1 to 10 o C in the dark? 7.7.1 7.9.1 3.19.1 Method Procedure 5.5 The volume of working DAPI applied and the incubation time ? 14.6 14.6 - Method Procedure 5.6 The technique used to drain the excess stain from the well and to rinse the well? 14.5 14.5 - Method Procedure 5.7 Wh at type and amount of mounting media used? 7.8 7.10 - Method Procedure

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  5.8 That all the edges of the cover slip are sealed well with clear fingernail polish, unless Elvanol ® is used? 14.9 14.9 - Method Procedure 5.9 The finished slides or slides not read immediately are stored in a humid chamber in the dark at 1 o to 10 o C (humid chamber not required for Elvanol ® )? 14.10 14.10 5.3.6 Method Procedure 5.10 Acceptable sample staining procedures, including issues not noted in items 5.1 through 5.9? Critical GLP **6 Microscope**  **and Examination** 6.1 Instructions for ocular and Kohler adjustments? 10.3.4 10.3.6 10.7

10.8 3.22.10 Requirement 6.2 That all measurements must be recorded to the nearest 0.5 micron? 15.2.2.3 15.2.3.3 15.2.2.4

15.2.3.4 3.22.5 Requirement 6.3 Microscope cleaning procedures? 10.4 10.9 3.22.11 Requirement 6.4 The recording of coordinates of all cysts and oocysts on the worksheet for future reference; and slide orientation on the microscope stage to standardize coordinate recording? - - - Recommendation
6.5 The examination and acceptance of positive and negative staining controls before proceeding with examination of field samples? 15.2.1 15.2.1 5.4.6 5.4.7 Requirement 6.6 That each analyst characterizes 3 oocysts and 3 cysts on the positive staining control at each examination session? 15.2.1.1 15.2.1.1 5.4.6 Requirement 6.7 Corrective actions if positive and/or negative staining controls are not acceptable? - - 5.4.8 Recommendation 6.8 The criteria for organism identification ? 15.2.2 15.2.2 15.2.3 5.4.9

5.4.10 Requirement 6.9 Every positive organism in a field sample is characterized and recorded? 15.2 15.2.2.1 15.2.3.1 5.4.9.1

5.4.10.1 Requirement 6.10 Acceptable microscope and examination procedures, including issues not noted in items 6.1 through 6. 9 ? Requirement GLP **7 Reagents**

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  7.1 Procedures for the preparation of all essential chemicals and reagents? 7.0 7.0 4.2 Critical 7.2 That expiration dates are specified for all reagents prepared by the laboratory? - - 4.2.2 Critical **8 Quality**

**A ssurance**  8.1 Training protocol for new employees? 9.1 9.1 1.7 Requirement GLP 8.2 Procedures for performing analyst verification? 10.6 9.10 7.1.9 Requirement GLP 8.3 Positive and interfering organisms detected in field samples are documented by photography? - - 5.4.11 Recommendation 8.4 Acceptable procedures for sample collection for field or utility personnel? - - 6.1 Critical GLP 8.5 Criteria for sample acceptance and corrective action procedures? 8.1.3 8.1.3 6. Requirement GLP 8.6 Method required holding times? 8.2 8.2 6.4 Requirement GLP 8.7 Manual data recording procedures? - - 8.0 Critical GLP 8. 8 Procedures for checking the accuracy of data transcriptions, including electronic data entry? - - 8.1 Critical GLP 8.9 Procedures for checking the accuracy of manual calculations? - - 8.1 Critical GLP 8.10 Procedures for electronic data entry and storage? - - 8.2 Critical GLP 8.11 How backup of stored data is performed? - - 8.2 Critical GLP 8.12 Corrective action procedures for OPR failures? 9.7.4 9.8.5 7.1.6.2 Requirement GLP 8.13 Corrective action procedures for method blank contamination? 9.6.2 9.7.3 7.1.5.2 Requirement GLP

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**Item to be Evaluated For each item, does the SOP specify:**  

### Reference* **Classification Satisfactory**  **Comments/ Response**  **Requested 1623**  **1623.1 Cert**  **Yes No**  **NA UNK**  8.14 Procedures for identifying and assessing declining trends in recovery through review of control charts and/or other recovery data? - - 7.1.7.2 Recommendation GLP 8.1 5 Corrective action procedures for investigating QC failures or declining trends in recovery? - - 7.1.7.2 Recommendation GLP 8.16 Acceptable glassware washing procedures? - - 4.4 Critical GLP 

### Comments:

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## Checklist **C –**  **Method 1623/1623.1 Technical Review –**  **Sample Processing and Microscopy Laboratory** 

**Name Name and A**  **ffiliation of Evaluator Date of Evaluation** 

Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the con sistency and reliability of results. Adapted from other federal programs for the purposes of the Cryptosporidium Laboratory QA Eva luation Program, GLP includes personnel, equipment, and standard operating procedures appropriate for the program. 

### Item to be evaluated **Reference*** **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK 1**  **Laboratory**

**Facilities** 1.1 Does laboratory appear to have established appropriate safety and health practices prior to use of this method? 5.0 5.0 4.1 Critical 1.2 Do all laboratory personnel wear gloves when handling biohazard and toxic compounds, and change gloves before touching other surfaces and equipment? 5.3 5.4 5.3 4.1.6 Critical GLP 1.3 Does the laboratory disinfect bench surfaces before and after analyses? - - 4.1.3 Critical GLP 1.4 Does the laboratory have adequate bench space to perform the method? - - 2.0 Critical GLP 1.5 Other than the issues noted in items 1.1 through 1.4 (if any), no other facility issues were observed? **2 Reagents** 2.1 Is reagent water used to prepare all reagents? 7.3 7.3 4.3.1 Requirement 2.2 Are all reagents clearly labeled with i dentity of reagent, date of preparation, technician initials, and expiration date? - - 4.2.2 Critical GLP

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 2.3 Are SOPs available in the work area, and does laboratory practice reflect written procedures? Critical GLP **3 Sample Spiking Technician:**  3.1 Wa s spike suspension vial vortexed for 30 seconds or per manufacturer’s instructions ? 11.4.3.1.2 11.2.3.2 - Method Procedure 3.2 Is the carboy used for method blank randomly selected from carboy stock to check efficacy of cleaning system? - - 7.1.5.3 Critical GLP 3.3 Wa s the suspension vial adequately rinsed? 11.4.3.1 11.2.3 - Method Procedure 3.4 Are SOPs for sample spiking available in the work area, and does laboratory practice reflect written procedures? Critical GLP 3.5 Other than issues noted for items 3.1 through 3.4 (if any) was sample spiking demonstrated successfully? **4 Filtration/Elution 4.1**  **Envirochek** ® HV **filtration Technician:**  4.1.1 Are all components required for sample filtration present and in good condition? 6.1 6.2.1 - 6.2.2 6.3 6.1 -

6.2.8 6.1.7 Requirement GLP 4.1.2 Is the filter assembly set up correctly? Figure 3a Figure 1 - Method Procedure GLP 4.1.3 Is the pump adequate for needs? 6.3.3 6.2.4 - Requirement GLP 4.1.4 Is the appropriate flow rate ma intained (approximately 2 L/ min)? 12.2.1.2 12.2.1.2 - Method Procedure 4.1.5 Is the volume filtered measured using a flow t otalizer or calibrated carboy? 12.2.4.2 12.2.4.2 - Requirement

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 4.1.6 Is the system well maintained and cleaned appropriately following use? 4.5 4.5 6.1.7 Critical GLP 4.1.7 Is the system able to maintain seal during use with no leaks? - - 6.1.7 Requirement GLP 4.1.8 Are SOPs for Envirochek® HV filtration available in the work area, and does laboratory practice reflect written procedures? Critical GLP
4.1. 9 Other than issues noted for items 4.1.1 through 4.1.8 (if any) was Envirochek® HV filtratio n demonstrated successfully? **4.2 Envirochek** ® **HV capsule filter elution**  **Technician:** 4.2.1 Is the elution buffer prepared as per Method ? 7.4.1 7.6.1 - Method Procedure 4.2.2 Is the wrist - shaker assembly set up correctly with arms fully extended ? 12.2.6.1.1 12.2.6.1 3.14.2 Method Procedure GLP 4.2.3 Is the dispersant addition performed as per Method 1623.1? - 12.2.7 - 1623 Recommendation **1623.1 Method**  **Procedure** 4.2.4 Is volume of elution buffer measured to ensure the use of one 250 mL centrifuge tube ? 12.2.6.2.2 12.2.8.2 - Method Procedure 4.2.5 Are the samples shaken at an appropriate speed ? 12.2.6.2.3 12.2.8.3 3.14.3 Method Procedure 4.2.6 Are the samples shaken three times for 5 minutes each time, and each in a different orientation? 12.2.6.2 12.2.8 - Method Procedure

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 4.2.7 Are SOPs for Envirochek® HV capsule filter elution available in the work area, and does laboratory practice reflect written procedures? Critical GLP
4.2.8 Other than issues noted for items 4.2.1 through 4.2.7 (if any) was Envirochek® HV capsule filter elution demonstrated successfully? **4.3 Filta**  **- Max** **® filtration**  **Technician:** 4.3.1 Wh ich filter is used

– Filta - Max ® (black end caps) or Filta - Max xp ress ® (red end caps)? 4.3.2 Are all components required for sample filtration present and in good condition? 6.1 6.2.1 6.2.3 6.3 6.1

6.2.1 - 6.2.7 6.2.9 6.1.7 Requirement GLP
4.3.3 Is the filter assembly set up correctly? Figure 3b Figure 2 - Method Procedure GLP 4.3.4 Is appropriate flow rate ma intained of <4 L per minute for Filta - Max

® ? 12.3.1.1.3 12.3.1.1.3 - Method Procedure 4.3.5 Is the volume filtered measured correctly using a flow meter or calibrated carboy? 12.3.1.5.2 12.3.1.5.2 - Requirement GLP 4.3.6 Is system well maintained and cleaned appropriately following use ? 12.3.4 12.3.4 6.1.7 Requirement GLP 4.3.7 Is system able to maintain seal during use with no leaks? - - 6.1.7 Requirement GLP 4.3.8 Does the laboratory indicate on the filter housing the correct direction of flow? 12.3.1.3 12.3.1.3 - Critical

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 4.3.9 Are SOPs for Filta - Max ® filtration available in the work area, and does laboratory practice reflect written procedures? Critical GLP 4.3. 10 Other than issues noted for items 4.3.1 through 4.3.9 (if any) was Filta - Max

® filtration demonstrated successfully? **4.4 Filta**  **- Max** ® **filter wash station elution Technician:**  4.4.1 Is an automatic or manual wash station used? 4.4.2 Is the filter wash station set up correctly? 12.3.2.1 12.3.2.1 - Requirement GLP 4.4. 3 Is residual suspension rinsed from all containers? 12.3.2.2.1d 12.3.2.2.1d - Critical 4.4. 4 Is PBST used to elute the filter? 7.4.2.4 7.6.2.4 - Method Procedure 4.4.5 Is an appropriate amount of PBST used for each wash? (approx. 600 mL) 12.3.2.2 12.3.2.2 - Method Procedure 4.4.6 During the first wash, is the plunger moved up and down 20 times? 12.3.2.2.1h 12.3.2.2.1h - Method Procedure 4.4.7 Is the plunger moved up and down gently to avoid generating excess foam? 12.3.2.2.1h 12.3.2.2.1h - M ethod Procedure 4.4. 8 During the second wash, is the plunger moved up and down 10 times ? 12.3.2.2.2b 12.3.2.2.2b - Method Procedure 4.4.9 If the automatic washer is used, is the machine operating properly? 12.3.2.1 12.3.2.1 - Requirement 4.4.10 Is the wash station cleaned adequately between samples ? 12.3.4.2 12.3.4.2 - Requirement GLP

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 4.4.11 Are SOPs for Filta - Max ® filter wash station elution available in the work area, and does laboratory practice reflect written procedures? Critical GLP
4.4.12 Other than issues noted for items 4.4.1 through 4.4.11 (if any) was Filta - Max

® filter wash station elution demonstrated successfully? **5 Concentration**  **5.1 Filta**  **- Max** ® **filter sample concentration Technician:**  5.1.1 Is concentrator set up correctly ? 12.3.3.2.1b 12.3.3.2.1a - Requirement GLP 5.1.2 Is the force of the vacuum ma intained below 30 cm Hg? NOTE pg 43 NOTE

pg 34 - Method Procedure 5.1.3 Is concentration performed after each of the washes? 12.3.2.2.1j 12.3.2.2.1j - Method Procedure 5.1.4 Is the sample concentrated so that some liquid remains above the filter ( enough to cover the stir bar about half-way)? 12.3.3.2.1c 12.3.3.2.1b - Method Procedure
5.1.5 Are the stir bar and concentration tube rinsed after each concentration and the liquid added to the concentrate? 12.3.3.2.1c 12.3.3.2.1b - Requirement
5.1.6 Wa s the filter membrane washed twice with 5 mL of PBST ? 12.3.3.2.3 12.3.3.2.3 - Method Procedure 5.1. 7 Are SOPs for Filta - Max ® filter sample concentration available in the work area, and does laboratory practice reflect written procedures? Critical GLP

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 5.1. 8 Other than issues noted for items 5.1.1 through 5.1.7 (if any) was Filta - Max

® filter sample concentration demonstrated successfully? **5.2 Envirochek** ® **HV and Filta**  **- Max** ® **filter sample centrifugation Technician:**  5.2.1 Is the sample centrifuged at 1500 x G (maximum 2000 x G) using a swinging bucket rotor? 13.2.1 and NOTE pg 46 13.2.1

and NOTE pg 37 - Method Procedure GLP 5.2.2 Are the centrifuge tubes properly balanced prior to centrifugation? - 13.2.1 3.15.4 Critical 5.2.3 Does lab have easily accessible me thod for determining relative centrifugal force of centrifuges? - - 3.15.1 Critical GLP 5.2.4 Is the sample centrifuged for 15 minutes , with time beginning when centrifuge reaches desired speed ? 13.2.1 13.2.1 - Method Procedure
5.2. 5 Is the centrifuge slowly decelerated at the end without the brake? 13.2.1 13.2.1 - Method Procedure 5.2. 6 Is the pellet volume determined? 13.2.1 13.2.1 5.2.3 Requirement 5.2. 7 Is there a set of standards for comparison of pellet size? - - 5.2.3 Recommendation GLP 5.2. 8 Are SOPs for Envirochek® and Filta - Max

® filter sample centrifugation available in the work area, and does laboratory practice reflect written procedures? Critical GLP
5.2. 9 Other than issues noted for items 5.2.1 through 5.2. 8 (if any) was Envirochek® HV or Filta - Max ® filter sample centrifugation demonstrated successfully?

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK 6**  **Purification and Slide Preparation Technician:**  6.1 Is an approved IMS kit/manufacturer used? 7.5 7.7.1 - Method Procedure GLP 6.2 Is the supernatant from the centrifuged sample aspirated no lower than 5 mL of supernatant above every 0.5 mL pellet or portion of 0.5 mL pellet ? 13.2.2 13.2.2 13.2.3 5.2.2

5.2.3 Requirement
6.2.1 Are the samples aspirated using the pipette, with the documented internal diameter, as specified in the SOP? - NOTE pg 37 - Critical
6.2.2 Is the proper rate (mL/min) or pressure (psi) maintained throughout aspiration? - 13.2.2 13.2.3 - Method Procedure
6.3 Is the pellet vortexed a sufficient time for resuspension ? 13.2.3 13.2.4.1.3 13.2.4.2 13.2.2.1

13.2.3.1.2 13.2.3.2 - Method Procedure 6.4 Is the resuspended pellet volume quantitatively transferred to the flat- sided tube (2 rinses )? 13.3.2.1 13.3.2.1 - Method Procedure 6.5 Are the IMS beads thoroughly resuspended prior to addition to the flat sided tube ? 13.3.2.2 13.3.2.4 13.3.2.2

13.3.2.4 - Method Procedure 6.6 Is the flat - sided tube rotated at 18 rpm for 1 hour at room temperature ? 13.3.2.6 13.3.2.6 - Method Procedure 6.7 Is the rotating mixer calibrated annually? - - 3.17.4 Critical GLP 6.8 Is flat - sided tube correctly placed in magnet and rocked through 90 degrees about once per second? 13.3.2.7 - 13.3.2.9 13.3.2.7 -

13.3.2.9 - Method Procedure 6.9 Is all the liquid removed when decanting is performed with the magnet up ? 13.3.2.11 13.3.2.11 - Method Procedure 6.10 Is the sample quantitatively transferred from the flat - sided tube to the microcentrifuge tube (2 rinses)? 13.3.2.13 13.3.2.14 - Method Procedure

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK**
6.11 Are the beads rinsed with PBS while inside the microcentrifuge tube? 13.3.4 13.3.2.17 - 1623 Recommendation 1623.1 Requirement 6.1 2 Is standard NaOH (5 μL, 1N) and standard HCl (50 μL, 0.1N) used? NOTE pg 49 & 50 NOTE

pg 42 3.17.5 Requirement GLP 6.1 3 Is sample vortexed vigorously for 50 seconds immediately after the addition of acid and 30 seconds after the sample has set for 10 minutes at room temperature? 13.3.3.2 - 13.3.3.4 13.3.3.2 -

13.3.3.4 - Method Procedure

6.1 4 Is a second dissociation performed ? 13.3.3.10 NOTE pg 49 13.3.3.10

NOTE pg 41 5.2.4 Requirement 6.1 5 Wh en the second dissociation is performed, does the laboratory: (A) use a second slide (B) add the additional volume to the original slide? 13.3.3.10 13.3.3.10 13.4.5 - Circle one: A B
6.1 6 Are the slides clearly labeled so they can be associated with the correct sample ? 13.3.3.7 13.3.3.7 - Requirement 6.1 7 Wh at type of slides is used? GLP 6.1 8 Is slide dried at: (A) room temperature, (B) 35o to 42oC, or (C) in the refrigerator? 13.3.3.12 13.3.3.12 - Circle one: A B C 6. 19 If the slide is wa rmed, is incubator or slide warmer calibrated and labeled? - - 3.4 Critical GLP 6.2 0 Are SOPs available in the work area for sample purification and slide preparation, and does laboratory practice reflect written procedures? Critical GLP 6.2 1 Other than issues noted for items 6.1 through 6.20 (if any) was p uri fication and slide preparation demonstrated successfully? **7 Sample Staining Technician:**  7.1 Wh at staining kit/manufacturer is used ? 14.2 14.2 3.18.1 GLP

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 7.2 Is FITC stain applied according to manufacturer’s directions ? 14.2 14.2 5.3.2 Method Procedure 7.3 Are positive and negative staining controls performed? 14.1 14.1 5.3.5 Requirement 7.4 Are the slides incubated in a humid chamber in the dark at room temperature for approximately 30 minutes or per manufacturer’s directions ? 14.3 14.3 5.3.3 Method Procedure
7.5 Are the labeling reagents rinsed away properly after incubation, without disturbing the sample? 14.5 14.5 - Method Procedure 7.6 Wa s the working DAPI stain prepared the day it was used ? 7.7.2 7.9.2 3.19.2 Method Procedure 7.7 Is stock DAPI stored at 1 to 10 o C in the dark? 7.7.1 7.9.1 3.19.1 Method Procedure 7.8 Is the DAPI stain applied properly and allowed to stand for a minimum of 1 minute? 14.6 14.6 - Method Procedure 7.9 Is the DAPI stain rinsed away properly without disturbing the sample? 14.7 14.7 - Method Procedure 7.10 Is the mounting media applied properly? 14.8 14.8 - Method Procedure 7.10.1 Wh at type of mounting media is used? 7.8 7.10 - GLP 7.10.2 Are all the edges of the cover slip sealed well with clea r fingernail polish, unless Elvanol ® is used? 7.9 14.9 7.11

14.9 - Method Procedure 7.11 Are the finished slides stored in a humid chamber in the dark at 1 to 10 o C (humid chamber not required for Elvanol ® )? 14.10 14.10 5.3.6 Method Procedure 7.12 Are SOPs for sample staining available in the work area, and does laboratory practice reflect written procedures? Critical GLP 7.13 Other than issues noted for items 7.1 through 7.12 (if any) was s ample staining demonstrated successfully?

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK 8**  **Microscope and Examination**  8.1 Is microscope equipped with appropriate excitation and band pass filters for examining FITC labeled specimens as demonstrated with lab, and auditor provided, positive staining control? 6.9.2 6.7.2 3.22.3 Requirement GLP
8.2 Is microscope equipped with appropriate excitation and band pass filters for examining DAPI labeled specimens as demonstrated with lab, and auditor provided, positive staining control? 6.9.3 6.7.3 3.22.3 Requirement GLP
8.3 Does the microscope have appropriate objectives and filters for DIC, which change easily to and from epifluorescence ? 6.9.1 6.7.1 3.22.4 Requirement GLP 8.4 Are all portions of the microscope, from the light sources to the oculars, properly adjusted? 10.3 10.0 Appendix B 3.22.6 Requirement 8. 5 Is the DIC image appropriate for each laboratory microscope ? - Figure 4 Visual Guide Requirement 8. 6 Is microscope cleaned after every session ? 10.4 10.9.8 3.22.11 Requirement GLP 8. 7 Does the microscope have a 20X scanning objective? 6.9.1 6.7.1 3.22.8 Requirement GLP 8. 8 Does the microscope have a 100X oil immersion objective? 6.9.1 6.7.1 3.22.8 Requirement GLP 8. 9 Is the microscope equipped with an ocular micrometer? 6.9.1 6.7.1 3.22.9 Requirement GLP 8. 10 Is a stage micrometer available to laboratory? 6.9.1 10.3.5 6.7.1

App. B 3 3.22.9 Requirement 8.1 1 Is a calibration table for 100X objective located close to the microscope(s )? 10.3.5.7 App. B 3.7 3.22.9 Requirement 8.1 2 Has the mercury bulb been used less than the maximum hours recommended by the manufacturer? 10.3.2.11 App.B 1.11 3.22.12 Requirement

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**Item to be evaluated Reference** * **Classification Satisfactory**  **Comments/ Response Requested**  **1623 1623.1**  **Cert Yes**  **No NA**  **UNK** 8.1 3 Does the laboratory have a preventative ma intenance agreement in place to service the microscope annually? - - 3.22.6 Critical GLP 8.1 4 Are SOPs for sample examination available in the work area, and does laboratory practice reflect written procedures? Critical GLP 8.15 Other than issues noted for items 8.1 through 8.13 (if any) was Microscope and Examination demonstrated successfully?

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- 37 **9 Positive Staining Control and OPR Slide**  **s** 9.1 Does the laboratory’s positive staining control slide contain ( oo ) cysts at the appropriate fluorescence intensity for FITC? 15.2.1.3 15.2.1.3 5.4.8 5.4.9.2 5.4.10.2 Requirement
9.2 Does the laboratory’s positive staining control slide contain ( oo ) cysts at the appropriate fluorescence intensity for DAPI? 15.2.1.3 15.2.1.3 5.4.8 5.4.9.3 5.4.10.3 Requirement
9.3 Does the laboratory’s positive staining control slide contain an appropriate level of background fluorescence? - - 5.4.3 Recommendation 9.4 Is concentration of oocysts on the positive staining control slide appropriate? 14.1.1 15.2.1.3 14.1.1

15.2.1.3 7.1.8.1 Requirement 9.5 Does the laboratory’s positive staining control exhibit appropriate contrast and organism features by DIC? - Figure 4 Visual Guide Requirement 9. 6 Does the laboratory’s OPR slide contain ( oo ) cysts at the appropriate fluorescence intensity for FITC? 15.2.2.1 15.2.3.1 15.2.2.2

15.2.3.2 5.4.9.2

5.4.10.2 Requirement 9. 7 Does the laboratory’s OPR slide contain ( oo ) cysts at the appropriate fluorescence intensity for DAPI? 15.2.2.2 15.2.3.2 15.2.2.3

15.2.3.3 5.4.9.3

5.4.10.3 Recommendation 9. 8 Does the laboratory’s OPR slide contain an appropriate level of background fluorescence? - - 5.4.3 Requirement 9.9 Does the laboratory’s OPR slide exhibit appropriate contrast and organism features by DIC? 9.7.1.1 9.8.1.1 Figure 4 Visual

Guide Requirement 9. 10 Does the technical auditor’s count of Cryptosporidium oocysts and Giardia cysts on the OPR slide sent by the laboratory agree within 10% of laboratory count? 10.6.3.1 9.10.3.1 7.1.9.4 Requirement 

### Comments:

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- 38 **1 0**  **Onsite Sample Processing** 

**Method Step Name**  **Position Demonstrated Technique**  **Successfully** yes/no Spiking

– (filter type) Filtration - (filter type) Spiking flat - sided tube, and processing IMS control Aspiration and transfer from 250 mL bottle

**11 Onsite Blind Spike**  **Results Sample**  **Cry pto Spike**  **Value Cry**  **pto Count Cry**  **pto Recovery** 

**(%) Giardia**  **Spike**

**Value Giardia**  **Count Giardia**  **Recovery**

**(%)**

**12 Evaluation of Onsite Sample Processing and Blind Spike Results**  **– Comments and Recommendations** 

**Classification Comments**  **Response Requested**

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- 39 **1 3**  **Was analy st microscope operation acceptable? (y** **es/no) Requirement**  Method 1623: 10.3.4 .1 Method 1623.1: 10.7.1 **Requirement** Method 1623: 10.3.4.2 - 3 Method 1623.1: 10.7.2 - 3 **Requirement** Method 1623: 10.3.6 Method 1623.1: 10.8 **Name Position**  **Ad just Interpupillary**  **Distance Focus both ey**  **epieces Establish Kohler Illumination** 

**14 Slide Count and Analy** **st Verification Results (y es/no)**  **Requirement** Method 1623: 10. 6.3.1 Method 1623.1: 9.10.3.1 **Requirement**

Method 1623: 10.6.3.1 Method 1623.1: 9.10.3.1 **Requirement**

Method 1623: 15.2 Method 1623.1: 15.2 **Requirement** Method 1623: 15.2.2.3 15.2.3.3 Method 1623.1: 15.2.2.4 15.2.3.4 **Requirement**

Method 1623: 15.2.2.3 15 .2.3.3 Method 1623.1: 15.2.2.4 15.2.3.4 **An al**  **yst Cry**  **pto Count Within**

**10% of Target Count Giardia**  **Count Within 10% of**  **Target Count Examine and Record**  **Characteristics Measurement (100X)**  **Demonstrated Internal Structures** 

**15 Evaluation**  **of Analy st Microscopy**  **and Examination Skills –**  **Comments and Recommendations Classification**  **Comments Response Requested** 

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# APPENDIX 

# H Positive Staining Control and OPR Slide Evaluation

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**Laboratory Name: Date:** 
**Technical A uditor:**  **Organism/Slide ID:** Cryptosporidium **1 2**  **3 4**  **Score**

**Positive Stain Control**  **Number of**

**Organisms [Sections 14.1.1 &**  **15.2.1.3 (Sections**  **14.1.1 & 15.2.1.3) ]**  organisms in very low numbers (<~25 oo/cysts) organisms in low numbers (~25 to ~100 oo/cysts) organisms in relatively high numbers(~100 to <~200 oo/cysts) organisms in high numbers (>~200 oo/cysts)
**FITC Fluorescence*** **[Section 15.2.1.3 (Section 15.2.1.3)**  **]** consistently weak (1+); inadequate; not crisp inconsistent; more weak (1+) than strong (3+); less than adequate; more not crisp than crisp inconsistent; more strong (3+) than weak (1+); adequate; more crisp than not crisp consistently strong (3+); superior; crisp
**DAPI Fluorescence  ** [Section 15.2.1.3** **(Section 15.2.1.3) ]**  DAPI negative in majority of oocysts; nuclei stained consistently weak (1+); inadequate distinct nuclei in less than half of the oocysts; nuclei stain inconsistent; more weak (1+) than strong (3+); less than adequate distinct nuclei stained in majority of oocysts; nuclei stain inconsistent; more strong (3+) than weak (1+); adequate distinct nuclei stained in majority of oocysts; nuclei stain cons istently strong (3+); superior **Background Fluorescence**  excessive, interfering distracting exists but does not interfere minimal to nonexistent **Background and Organism**  **Characteristics on DIC**  interfering; atypical distracting; atypical exists but does not interfere; typical minimal to nonexistent; typical

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**OPR Number of** 

**Organisms [Section 10.6.3.1**  **(Section 9.10.3.10) ]**  difference in number counted >20% 16 - 20% difference 10 - 15% difference difference in number counted <10% **FITC Fluorescence** * **[Section 15.2.2.1 (Section 15.2.2.2)**  **]** consistently weak (1+); inadequate; not crisp inconsistent; more weak (1+) than strong (3+); less than adequate; more not crisp than crisp inconsistent; more strong (3+) than weak (1+); adequate; more crisp than not crisp consistently strong (3+); superior; crisp
**DAPI Fluorescence  ** [Section 15.2.2.2** **(Section 15.2.2.3) ]**  DAPI negative in majority of oocysts; nuclei stained consistently weak (1+), inadequate distinct nuclei in less than half of the oocysts; nuclei stain inconsistent; more weak (1+) than strong (3+); less than adequate distinct nuclei stained in majority of oocysts; nuclei stain inconsistent; more strong (3+) than weak (1+); adequate distinct nuclei stained in majority of oocysts; n uclei stain consistently strong (3+), superior **Background Fluorescence**  excessive, interfering distracting exists but does not interfere minimal to nonexistent **Background and Organism**  **Characteristics on DIC**  **[ (Figure 4)**  **]** interfering; <50% of oo/cysts undamaged and morphologically intact distracting; <50% oo/cysts undamaged and morphologically intact exists but does not interfere; >50% oo/cysts undamaged and morphologically intact minimal to nonexistent; >50% oo/cysts undamaged and morphologically int act

*FITC Fluorescence: brilliant apple - green ovoid or spherical objects with brightly highlighted edges (referred to as crisp above); compared for all organisms ove r complete well slide at 200x **DAPI Fluorescence: light blue internal staining (no distinct nuclei) with green rim, intense blue internal staining, or distinct, sky- blue nuclei; compared for all organisms over complete well slide at minimum of 400x Fluorescence intensity scale: 1+ = weak, 2+=medium, 3+=strong For visual co mparisons, see FITC and DAPI staining examples in Method 1623.1 Microscopy Visual Guide (p. 61 in Method 1623.1) or the LT2 O n - line Microscopy Training Module (http://water.epa.gov/lawsregs/rulesregs/sdwa/lt2/compliance.cfm#training). For descriptions, see Method 1623/1623.1 Sections 15.2.2 and 15.2.3. Section references in [ ] refer to EPA Method 1623, and the corresponding section references in parentheses and italics refe r to EPA Method 1623.1. Satisfactory scores are ≥ 3. Any score ≤ 2 (shown in red font) indicates problems with slide preparation and should be invest igated. If slide evaluation is conducted as part of the Assessment of Cryptosporidium Laboratory Quality Assurance under the Safe Drinking W ater Act, scores, comments and any requirements, recommendations, or commendations should be included in Section 9 of Checklist C - Method 1623/1623.1 Technical Review - Sample Processing and Microscop y.
**Comments:** PSC:

OPR: **Requirements, Recommendations,**  **Commendations:**

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# APPENDIX 

# I Example Report from Online Analy st Evaluation

### I
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Numbe r Que sti on Val ue Ana lyst 1 1 I (i nse rt name )< 1> h ave comp let ed al l the n ec essary trai ni ngt o be an anal yst f or M et hods 1623 an d 1623. 1. I am compl et in g thi s anal yst ver ific ati on wi thout assi stanc e fr om othe rs on the d ate l is te d ( mm/dd/yea r) <2>. 0 0 2 Have t he la boratory QA of fic ercompl et e the f ol lo wingp hrase : <1>co nfir m that anal yst ( in se rt anal yst name ) <2>co mpl et ed thi s ev al uati on wit hout assi stanc e on (m m/dd/year) <3> 0 0 3 De sc ri be t he FITC of Organi sm 1. ( Se lect al l that appl y.) 5 5 4 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 1 ? 10 10 5 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 1? 10 10 6 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 1 ? ( Se lect al l that appl y.) 5 5 7 Wh at woul d you cal l Organi sm 1 ? 10 10 8 De sc ri be t he FITC of Organi sm 2. ( Se lect al l that appl y.) 5 5 9 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 2 ? 10 10 10 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 2? 10 10 11 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 2 ? ( Se lect al l that appl y.) 5 5 12 Wh at woul d you cal l Organi sm 2 ? 10 10 13 De sc ri be t he FITC of Organi sm 3. ( Se lect al l that appl y.) 5 5 14 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 3 ? 10 10 15 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 3? 10 0 16 Wh at in te rnal structure s, if any, di d you observe f or Organi sm 3? ( Select al l that appl y.) 5 0 17 Wh at woul d you cal l Organi sm 3 ? 10 0 18 De sc ri be t he FITC of Organi sm 4. ( Select al l that appl y.) 5 0 19 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 4 ? 10 10 20 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 4? 10 10 21 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 4 ? ( Se lect al l that appl y.) 10 10 22 Wh at woul d you cal l Organi sm 4 ? 10 10 23 De sc ri be t he FITC of Organi sm 5. ( Se lect al l that appl y.) 5 5 24 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 5 ? 10 10 25 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 5? 10 10 26 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 5 ? (Se lect al l th at ap pl y.) 5 0 27 Wh at woul d you cal l Organi sm 5 ? 10 10 28 De sc ri be t he FITC of Organi sm 6. ( Select al l that appl y.) 5 5 29 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 6 ? 10 10 30 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 6? 10 10 31 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 6 ? ( Se lect al l that appl y.) 5 5 32 Wh at woul d you cal l Organi sm 6 ? 10 0 33 De sc ri be t he FITC of Organi sm 7. ( Se lect al l that appl y.) 5 0 34 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 7 ? 10 0 35 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 7? 10 10 36 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 7 ? ( Se lect al l that appl y.) 10 10 37 Wh at woul d you cal l Organi sm 7 ? 10 10 38 De sc ri be t he FITC of Organi sm 8. ( Se lect al l that appl y.) 5 5 39 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 8 ? 10 10 40 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 8? 10 0 41 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 8 ? ( Se lect al l that appl y.) 10 10 42 Wh at woul d you cal l Organi sm 8 ? 10 0 43 De sc ri be t he FITC of Organi sm 9. ( Select al l that appl y.) 5 5 44 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 9 ? 10 0 45 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 9? 10 10 46 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 9 ? ( Se lect al l that appl y.) 10 10 47 Wh at woul d you cal l Organi sm 9 ? 10 10 48 De sc ri be t he FITC of Organi sm 10. ( Se lect al l that appl y.) 5 0 49 Wh at woul d you re co rd on y our slide exa mi nati on for m for the DA PI stai ni ng of Organi sm 1 0? 10 10 50 Wh at woul d you re co rd on y our slide exa mi nati on for m base d on the DI C of Organi sm 10? 10 10 51 Wh at in te rnal structure s, if any, di d you observe f or Organ is m 1 0? ( Se lect al l that appl y.) 5 5 52 Wh at woul d you cal l Organi sm 1 0? 10 10 To tal 420 325 Per ce ntage 77

### J
- 1 

# APPENDIX 

# J Method 1623 Ongoing Precision and Recovery (OPR) and Initial Precision and Recovery (IPR) Quality Control Criteria Calculated from Five Rounds of Proficiency Testing (PT) Data

### J
- 2

**Method 1623 Ongoing Precision and Recover y (OPR) and Initial Precision and**  **Recovery (IPR) Quality Control Criteria Calculated from**  **Five Rounds of**

**Proficiency**

**Testing (PT)**  **Data Updated**  **July**

**9, 2013 Introduction**  The December 2005 Method 1623 Ongoing Precision and Recovery (OPR) and Initial Precision and Recovery

(IPR) quality

control and acceptance criteria were developed based on 293 Cryptosporidium OPR samples and 186 Giardia OPR samples analy zed by six laborato ries during the Information Collection Rule Supplemental Survey s from March 1999 to February 2000. In 2009, the minimum Cryptosporidium percent recovery for OPR samples was updated based on anal ys is of data from the February and May 2007 proficiency

testing (PT) rounds as published in the Federal Register ( Federal Register Notice, Vol. 74, No. 36 February 25, 2009 )
. To more accurately

reflect current laboratory capability

and provide better values for assessment of laboratory

performance, data from five PT rounds (February and May 2007, July

2009, February 2010, and March 2011) were used to develop quality control criteria. For each round, laboratories anal yz ed three blind reagent water samples spiked with flow
- cy tometer counted organisms prepared b y the Wisconsin State L aboratory of Hygiene. These updated criteria provide a better assessment of laboratory capability

than the 2009 values for the following reasons: 1) the data set is more recent; 2) the sample size is more than twice as large as the 2009 sample size; and 3) the updated value of 33% for Cryptosporidium OPR samples is consistent with the Quality

Control Acceptance Criteria developed with Method 1623.1 ( EPA Method 1623.1, January

2012 (PDF) (83 pp, 1MB). A summary of the data set and proposed acceptance criteria are shown in Table 1. **Calculation of OPR Minimum Recovery Criteria**  The minimum Cryptosporidium percent recovery criteria for OPR samples were calculated based on data from the February and May 2007, July 2009, February

2010, and March 2011 PT rounds, which were all of the PT rounds with protozoan suspensions in reagent water without any additional matrix material. The remaining PT rounds had matrix additions and would not simulate an OPR or IPR. The acceptance criteria were calculated using data from labs approved and participating in the Lab QA Program as of June 24, 2011. The cal culated lower limit of recovery was 33% for Cryptosporidium and 29% for Giardia . When applying the criteria to the data from the five PT rounds, 51 samples or 6.8 % of the 753 samples did not meet the criteri on for Cryptosporidium , and 31 samples, or 5.8% of the 530 samples, did not meet the criterion for Giardia
. **Details of the Calculation of OPR Minimum Recovery Criteria** To calculate OPR recovery criteria, estimates of variance attributable to four different sources were calculated: variability between laboratories, variability between PT rounds, variability between laboratory
- and
- round (i.e., attributable to an interactio n between round and laboratory), and variability

within round and laboratory (i.e., analy

tical variability). There were a few laboratories that performed PT anal ys es using multiple filters. For the purpose of these calculations, the two sets of anal ys es were two separate entities; in other words, the two lab/filter

### J
- 3

combinations were treated as two different laboratories. The different variance components were calculated using PROC MIXED from SAS version 8 using the maximum likelihood method of estimation on recovery

data. Details on the maximum likelihood estimation can be found in SAS/STAT User’s Guide. 1 Estimates of between laboratory variance, between round variance, between laboratory
- and
round variance, and within laboratory
- and
- round variance were labeled s 2 L 

### , s 2 R 

### , s 2 LR 

### and s 2 w 

### , respectivel y. The combined standard deviation (s c 

### ) is:
2 2 2 2 

### ) * * 1 1 ( ) * 1 1 ( ) 1 1 ( ) 1 1 ( w LR R L c 

### s n R L s R L s R s L s         

### Wh ere: 

### R C L  = average number of laboratory/filter combinations per round


### L C R  = average number of rounds per laboratory/filter combination n = average number of replicates per laboratory/filter and round (3 for Cry ptosporidium

, 2.2 for Giardia

) C = total number of laboratories/filters/rounds R = number of rounds (5) L = number of laboratory/filter combinations Lower recovery

limits for OPR samples were then calculated as:

c df Mean 

## s t X *

) , 95
. 0 ( 

##  Wh ere: X mean 

### = the mean recovery of all samples t (0.95,df) 

### = 95 th 

### percentile of the student’s t distribution with df degrees of freedom df is calcu lated using Satterthwaite’s estimate as given below:


### ) 1 ( * * ) ) * * 1 1 (( ) 1 )( 1 ( ) ) * 1 1 (( 1 ) ) 1 1 (( 1 ) ) 1 1 (( 2 2 2 2 2 2 2 2 4



###   

   

  

  

n R L s n R L R L s R L R s R L s L s df

w LR R L c



### Calculation of IPR Minimum Mean Recovery **and Maximum RSD Criteria** The QC acceptance criteria for IPR samples were calculated based on data from the February and May 2007, July

2009, February

2010, and March 2011 PT rounds. The calculated lower limit of recovery was 36% for Cryptosporidium and 32% for Giardia . When appl yi ng the criteria to the data from the five PT rounds, 16 (6.4%) of the 251 round/filter/laboratory combinations did not meet the criteri on for minimum mean recovery for Cryptosporidium , and 17 (7.1%) of the 240 combinations did not meet the minimum mean recovery criterion for Giardia . The maximum RSD of 34 % for Cryptosporidium and 37% for Giardia was also calculated using data from the five PT rounds. Applying the criteria to the data from all laboratories, 14 laboratory/ filter/round combinations (5.6%) did not meet the Cryptosporidium criterion and 14 combinations (5.8%) did not meet the Giardia criterion. Six of the 16 1 

### SAS Institute Inc. 1994. SAS/STAT User’s Guide, Volume 2, GLM
-VARCOMP. Version 6, 4 th 

### Edition, June 1994.

### J
- 4

Cryptosporidium lab/round/filter combinations and 5 of the 17 Giardia combinations that failed the lower recovery

criterion also failed the precision criterion. It is worth noting that because the criteria are calculated for 4 s amples, while the data included onl y 3 ( Cryptosporidium ) or 2 ( Giardia ) results per lab, the failure rates would be expected to be greater than the target 5%. However, due to the large number of laboratory /filter/round combinations, the failure rate was ≈ 5%. **Details of the Calculation of IPR Criteria** Similar to the calculations for the OPR recovery criteria, IPR recovery

criteria were calculated with estimates of variance attributable to four different sources: variability between laboratories, variability between PT rounds, variability between laboratory
- and
- round (i.e., attributable to an interaction between round and laboratory), and variability within round and laboratory (i.e., anal ytical variability). There were a few laboratories that performed PT analy ses using multiple filters. For the purpose of these calculations, the two sets of anal ys es were two separate entities; in other words, the two lab/filter combinations were treated as two different laboratories. The different variance components were calculated using PROC MIXED from SAS version 8 using the maximum likelihood method of estimation on recovery data. Details on the maximum likelihood estimation can be found in SAS/STAT User’s Guide. 2 Estimates of between laboratory variance, between roun d variance, between laboratory
- and
round variance, and within laboratory
- and
- round variance were labeled s 2 L 

### , s 2 R 

### , s 2 LR 

### and s 2 w 

### , respectivel y. The combined standard deviation (s c 

### ) is:
2 2 2 2 

### ) * * 1 4 1 ( ) * 1 1 ( ) 1 1 ( ) 1 1 ( w LR R L c 

### s n R L s R L s R s L s         

### Wh ere: 

### R C L  = average number of laboratory/filter combinations per round


### L C R  = average number o f rounds per laboratory/filter combination n = average number of replicates per laboratory/filter and round (3 for Cry ptosporidium

, 2.2 for Giardia

) C = total number of laboratories/filters/rounds R = number of rounds (5) L = number of laboratory/filter combinations Lower recovery

limits for the mean of IP R sample results were then calculated as:

c df Mean 

## s t X *

) , 95
. 0 ( 

##  Wh ere: X mean 

### = the mean recovery of all samples t (0.95,df) 

### = 95 th 

### percentile of the student’s t distribution with df degrees of freedom df is calculated using Satterthwaite’s estimate as given below:

2 

### SAS Institute Inc. 1994. SAS/STAT User’s Guide, Volume 2, GLM
-VARCOMP. Version 6, 4 th 

### Edition, June 1994.

### J
- 5 

### ) 1 ( * * ) ) * * 1 4 1 (( ) 1 )( 1 ( ) ) * 1 1 (( 1 ) ) 1 1 (( 1 ) ) 1 1 (( 2 2 2 2 2 2 2 2 4



###   

   

  

  

n R L s

n R L R L s R L R s R L s L s df

w LR R L c


### The precision criterion for IPR samples was calculated as a maximum relative standard deviation (RSD)
. The maximum RSD was determined b y first pooling the individual laboratory /round RSDs using the formula below: 

# 

 

###  C

i i pool 

### RSD C RSD 1 2



### 1 

### Wh ere: RSD i 

### = the RSD calculated for laboratory/filter/round i , and C = the number of laboratory/filter/round combinations

. The maximum RSD was then calculated as: Pool C n Max 

## RSD F RSD T 

## * ) , 3 ; 95
. 0 (

 

##  Wh ere: n T 

### = the total number of results from all laboratories over all rounds, and C = the number of laboratory/filter/round combinations
. **Table 1. Summary**

**of A cceptance Criteria**  **Me thod 1623**  **2005 version Federal Register**  **74:36 (February 25, 2009)**  **p. 8529 Updated Value**  OPR Criteria

– Lower Limit Cryptosporidium - 11% Giardia - 14% Cryptosporidium - 22% Cryptosporidium - 33% Giardia - 29% IPR Criteria

– Lower Limit Cryptosporidium - 24% Giardia - 24% NA Cryptosporidium - 36% Giardia - 32% IPR Criteria

– Precision (maximum RSD) Cryptosporidium - 55% Giardia - 49% NA Cryptosporidium - 34% Giardia - 37% Data Set ICRSS data generated in 1999 and 2000 PT data generated during the February and May 2007

rounds PT data generated, as of June

24, 2011, during five PT rounds Number of Laboratories/Filters 6 58 Cryptosporidium - 56 Giardia - 55 Number of Samples 293 333 Cryptosporidium - 753 Giardia - 530 Method Version Used 1999 version of Method 1623
– Envirochek only 2005 version of Method 1623
– all filters 2005 version of Method 1623

– all filters Blind vs. Unblind Unblind Blind Blind

### K
- 1 

# APPENDIX 

# K Burden Tables

### K
- 2

Com plet e and su bm it laborat ory ev

aluat ion applic at ion pac ka ge (Rpt , TPD- Sta rt up and O&M c os ts) 0 3 10 1 13 22. 5 293

$
- $ 257. 25

$ 799. 50

$ 48. 00

$ 1, 104. 75 $
-

$ $3, 920. 00 113, 056. 88 $ $24, 856. 88 88, 200. 00 $ 84. 71

$ Pe rf orm off-s ite ev aluation activ

ities (OP R/ PSC ev aluation, online analy st v eri fi ca tio n) (Rpt ) 0 4 18 0 22 22. 5 486

$
- $ 367. 50

$ 1, 435. 00

$ 24. 00

$ 1, 826. 50 $
-

$ $700. 00 56, 846. 25 $ $41, 096. 25 15, 750. 00 $ 84. 63

$ Hos t o n-s ite ev aluation (includes co unt ing slid es ) (Rpt ; TP D - O&M co sts) 0 14 24 2 40 22. 5 889

$
- $ 1, 323. 00

$ 1, 968. 00

$ 144. 00

$ 3, 435. 00 $
-

$ $3, 254. 00 150, 502. 50 $ $77, 287. 50 73, 215. 00 $ 86. 96

$ Pe rf orm and report 2 se ts of profic ienc y te sts ( PT) (Rpt ) 0 4 29 2 34 45 1541

- $ 343. 00

$ 2, 357. 50

$ 144. 00

$ 2, 844. 50 $
-

$ $2, 225. 00 228, 127. 50 $ $128, 002. 50 100, 125. 00 $ 83. 05

$ Pe rf orm fol low-up ac tio n for poor PT res ults ( Rpt ) 0 3 19 1 23 7 158

- $ 294. 00

$ 1, 517. 00

$ 72. 00

$ 1, 883. 00 $
-

$ $1, 266. 67 22, 047. 67 $ $13, 181. 00 8, 866. 67 $ 83. 69

$ Pe rf orm and report 2 se ts of PT

s f or eac h additional method ve rs ion (Rpt ) 0 4 29 2 34 4 137

- $ 343. 00

$ 2, 357. 50

$ 144. 00

$ 2, 844. 50 $
-

$ $2, 225. 00 20, 278. 00 $ $11, 378. 00 8, 900. 00 $ 83. 05

$ 98 82 72 **Tota l**  3504 590, 858. 79 $ $295, 802. 13 295, 056. 67 $

Rpt -Report ing RK-Rec ord ke eping TP D - Th ird-part y disclo su re O& M C os ts toPe rform off-s itee va lu at ion activ ities : slid e produc tion, cost of sh ipping s lides to States/EPA f or ev aluat ion **Tota l**  **cos t/**  **ye ar**  **Acti vi**  **ty La**  **bor Cos**  **ts Ca**  **pit al/St** **ar**

**t up**  **O& M C**  **os**

**ts Num**  **ber**

**of re**  **sp onde**  **nts**

**/y ear**  **Legal $65.**  **00/ hour**  **Ma nagement** 

**$98. 00/**  **hour Te**  **chnica l** 

**$82. 00/**  **hour Cl**  **er ic**  **al**

**$72. 00/**  **hour Re**  **sp onde**  **nt**

**Ho urs** 

Num ber of res pondent s/ye ar toPer form f ol low-up action f

or poor PT

r es ult s : Estima te d 7 laboratories w ill h av e to p erform f

ollow-up due top oor PT r es ul ts Num ber of res pondent s/ye ar toPer form a nd report 2se ts of PT s f or eac h addit ional method v ers ion: Estimated 4 laborat ories w ill p erform mo re th an one method v ers ion **Table 1.**  **P ar** 

**ticip ating L**  **aboratories Seeking Certification**

Num ber of res pondent s/ye ar toC omplete and submit audit package: Tot al of a pprox imately 45 laboratories seek ing approv al div id ed by two ye ars Num ber of res pondent s/ye ar toPer form off-s itee va luat ion ac tiv ities: T ot al of approx imately 45 laboratories se ek ing approv al div ided by two ye ars Num ber of res pondent s/ye ar toH ost o n-s it e ev aluation: T

otal of a pprox imately 45 laborat ories se eking approv al div ided by two years Num ber of res pondent s/ye ar toPer form a nd report 2se ts of profic ienc y tests ( PT): Ap prox imately 4 5 labs p art icip at ing in the Lab QA Program O& M C os ts toH os t o n-s ite ev aluat ion: co st of audit package including slides, trav el costs f

or auditor O& M C os ts toPe rform and report 2se ts of p rofi ciency te sts ( PT): co st of PT sa mp les a nd su pplies O& M C os ts toPe rform fol low-up ac tio n for poor PT r es ults : co st of

a ddit ional blind samples and s upplies O& M C os ts toPe rform and report 2se ts of PT s f or eac h additional method v ers ion: cost of

PT samp les and supplies **Labor Cos t/**  **Acti vi**  **ty O&M** 

**Cos t/** **Acti vi**  **ty La**  **bor**

**Cos t/**  **Hr**

O& M C os ts toC om plet e and su bm it la borat ory e va luat ion applic ation pac ka ge: a pplication f ee (av erage va lue from 2la b and 2 sta te s) **Tota l**  **hours /y** **ear**

### K
- 3 Rev iew laboratory ev aluat ion pac ka ges ( RK ) 16.5 2 18.5 22. 5 416 $ $1 ,7 65. 50 $240.00 $2 ,0 05. 50 - $ 45,123.75 $ $45,123.75
- $ Conduc t a nd rev iew of f-s ite ev aluat ion ac tiv ities ( Rpt ) 12 2 14 22. 5 315

$ $1 ,2 84. 00 $240.00 $1 ,5 24. 00 - $ 34,290.00 $ $34,290.00
- $ Conduc t a nd rev iew on-s ite ev aluat ions ( Rpt ) 18 2 20 22. 5 450

$ $1 ,9 26. 00 $240.00 $2 ,1 66. 00 - $ 48,735.00 $ $48,735.00
- $ Not if y laboratories of ce rtif ica tio n status and maintain certif

ied laboratorylist o n Sta tewebs ite (Rpt ) 25.5 2 27.5 22. 5 619

$ $2 ,7 28. 50 $240.00 $2 ,9 68. 50 - $ 66,791.25 $ $66,791.25
- $ Rev iew and track 2 se ts of PT res ults ( RK ) 3 3 45 135 $ $ 3 21. 00 $- $3 21.00 - $ 14,445.00 $ $14,445.00
- $ Coordinat e fol lowup ac tiv

ities f or poor PT

r es ults (RK ) 3 3 7 21 $ $ 3 21. 00 $- $3 21.00 - $ 2, 247.00 $ $2,247.00
- $ Rev iew and track 2 se ts of PT res ults f

or additional method ve rs ions (R K) 3 3 4 12
$ $ 3 21. 00 $- $3 21.00 - $ 1, 284.00 $ $1,284.00
- $ 107 120 **Tota l**  1968 212,916.00 $ $212, 916.00 -

O&M C os ts: n one

Rpt - Report ing RK- Record keeping TP D - Th ird-partyd isclo su re **Ta ble 2**  **. Stat e A**  **nnua l B**  **urde n**  **Acti vi**  **ty Ce**  **rti fi**  **ca ti**  **on Offi**  **ce r/**  **Te chnica**  **l $107/**  **hr St** **at es**  **hrs /y**  **r/res p**  **Labor**

**cos t/yr/re**  **sp Ca**  **pit al** 

**Sta rtup**  **Cos t**  **O&M**

**Cos ts**  **Num be**  **r of La**  **bs Tota**  **l**

**hrs /yr**  **Tota l C**  **os ts**  **pe r**  **Ye ar** 

Num ber of res pondent s/ye ar toRev iew and track PT r es ults ( PT

): Ap prox imately 45 labs partic ipating in the Lab QA Program Num ber of res pondent s/ye ar toC oordinat e fol low-up ac tiv ities f or poor PT

r es ults: Estimated 7 laboratories will hav e to perf

orm fol low-up due top oor PT r esults Num ber of res pondent s/ye ar toRev iew and track 2 sets of PT r esults f

or addit ional method v ers ions : Estimated 4 laboratories will be perf orm ing more than one method v ers ion **Management $120/**  **hr**

Informat ion f

or Legal, Cl eric al, a nd Expert/Contrac to r pers onnel ca tegories was n ot included because c ons ult at ions rev ealed t hos e pers onnel were nota pplicable. Num ber of res pondent s/ye ar toRev iew laborat ory e va luation pac ka ges : T ot al of approx imately 45 laboratories seek ing certif ication div

ided by two years Num ber of res pondent s/ye ar toC onduc t a nd rev iew off-s ite ev

aluation activ

ities: T

ot al of approx imately 45 laboratories seek ing certif ication div

ided by two years Num ber of res pondent s/ye ar toC onduc t a nd rev iew on-s ite ev

aluat ions : T

ot al of approx imately 45 laboratoriesse eking certif ic at ion div

ided by two ye ars Num ber of res pondent s/ye ar toN ot if y la borat ories of ce rtif ica tio n sta tus: T ot al of approx imately 45 laboratories seeking certif ication div

ided by two ye ars **Labor Cos**  **t/Activity O&M** 

**Cos t/Activity** 

### K
- 4

Assist State Cert if ication Offi cers ( CO) - la borat ory ev aluation application pack ages 5 0.5 15 20. 5 22. 5 461

$ $ 43 6.25 $ 3 1.30 $ 65 3.55 $ 1 ,121.10 - $ 25, 224. 75 $ $25, 224. 75 - $ Assist State Cert if ication Offi cers ( CO) - off

si teev al uat io n ac tiv ities (OPR/PSC ev aluation, online analys t ve ri fi ca ti on) 5 0.5 12 17. 5 22. 5 394

$ $ 43 6.25 $ 3 1.30 $ 52 2.84 $9 90.39 1, 775. 00 $ 24, 058. 78 $ $22, 283. 78 1, 775. 00 $ Assist State Cert if ication Offi cers ( CO) - onsi teev al uat io n ac tiv ities 10 0.5 8 18. 5 22. 5 416

$ $ 87 2.50 $ 3 1.30 $ 34 8.56 $ 1 ,252.36 - $ 28, 178. 10 $ $28, 178. 10 - $ Assist State Cert if ication Offi cers ( CO) - in te rpret at ion of PT res ul ts ( 2 se ts/ye ar) 7 0.25 4 11. 25 45. 0 506

$ $ 61 0.75 $ 1 5.65 $ 17 4.28 $8 00.68 - $ 36, 030. 60 $ $36, 030. 60 - $ Assist State Cert if ication Offi cers ( CO) - in te rpret at ion of PT res ul ts f

or additional method ve rs ions ( 2 se ts/ye ar) 7 0.25 4 11. 25 4.0 45

$ $ 61 0.75 $ 1 5.65 $ 17 4.28 $8 00.68 - $ 3, 202. 72 $ $3, 202. 72 - $ Maintain link s to Sta te 's w ebs ite s for lists of ce rt ified la borat ories 3 0.5 3 7 20. 0 130

$ $ 26 1.75 $ 3 1.30 $ 13 0.71 $4 23.76 - $ 8, 475. 20 $ $8, 475. 20 - $ 87. 25 62.6 43. 57 **Tota l**  1953 125, 170. 15 $ $123, 395. 15 $1, 775. 00 Informat ion f

or Legal, Clerical, a nd Expert/Cont rac tor personnel c at egories was n ot included becaus e cons ultations r ev ealed thos e pers onnel were not applic able.
O& M Cos ts: co st f or so ft ware licens e (A dobe Connec t)and annual microsc ope se rv ice **Ta ble**  **3 . A** **ge ncy Annua**  **l B urde**  **n Acti**  **vi ty**  **Progra m** 

**Ma na**  **gement GS 14**  **$87. 25/**  **hr Manage**  **me nt** 

**$62. 60/**  **hr Tec**  **hni ca**  **l**

**$43. 57/**  **hr Ag**  **en cy** 

**hrs /yr/re**  **sp Labor** 

**cos t/**  **yr/re sp**  **O&M Cos**  **t/ Acti**  **vi ty**  **La bor**  **Cos t/**  **Acti vi**  **ty**

Cos ts a nd hours r

ef le ct 4 5 labs se eking c ert if ication once ev

ery 2 ye ars (45/ 2=22.5), 45 labs co mpleting 2 PT r ounds p er ye ar, and 20 State s w ith ce rt ific at ion programs **Ca pit** **al Sta**  **rtup Co**  **st O&M** 

**Cos ts**  **Num be**  **r of La**  **bora torie**  **s Tota**  **l**

**hrs /yr**  **Tota l C**  **os**

**ts pe**  **r Y ear** 

### K
- 5 **Nu mb**  **er o**  **f re**  **sp onde**  **nt s**  **Nu mb**  **er o**  **f Acti**  **vi ti**  **es Tot** **al**

**hours /y** **ear Tot** **al L**  **ab or** 

**cos t/**  **ye ar**  **Tot al**  **A nnua**  **l**

**Ca pi**  **ta l c**  **os**

**ts Tot** **al A**  **nnua l**  **O&M Co**  **st s**  **Tot al** 

**An nua**  **lize d**  **Co st**  Laborat ori es 45 6 3504 295, 802. 13 $
-

$ 295, 056. 67

$ 590, 858. 79

$ Sta te s 20 7 1968 212, 916. 00 $
-

$
-

$ 212, 916. 00

$ **Tot al**  **B urde**  **n** 65 13 5472 508, 718. 13 $
-

$ 295, 056. 67

$ 803, 774. 79

$

**N um**  **be r**  **of re**  **sp onde**  **nt s**  **N um**  **be r** 

**of Acti**  **vi ti**  **es T**  **ot al** 

**hours /y** **ear T**  **ot al**  **L ab**  **or**

**cos t/**  **ye ar**  **T ot** **al**

**An nua**  **l Ca**  **pi ta**  **l c os** 

**ts T**  **ot al**  **A nnua**  **l O&M**  **Co st** **s Tot** **al**

**An nua**  **lize d**  **Co st**  **Tot al**  **B urde**  **n** 1 6 1953 123, 395. 15 $
-

$ 1, 775. 00

$ 125, 170. 15

$ **Ta bl**  **e 4b**  **. T ot** **al A** **ge nc** **y Bu**  **rde n**  **Ta bl**  **e 4a**  **. T ot** **al R** **es ponde**  **nt B**  **urde n**